Development of new Parameters for
StructureDetermination and Dynamic Investigations
on
Biomacromolecules by NMR
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MASSACHUSETTS INSTITUTE,
OF TECHNOLO",
MAR 2 5 2005
by
LIBRARIES
Elke Duchardt
Dipiphil mzt,Bidriste
Unirsity fFrankfut Gernd 1999
SUBMITtED TO THE DEPARTMENT OF CHEMISTRY IN PARTIAL FULFILL OF THE
REQUIREMENTS FOR THE DEGREE OF
DOCIOR OF PHILOSOPHY IN BIOLOGICAL CHEMISTRY
AT THE
MASSACHIUSETTSINSTITUTE OF TECHNOLOGY
Febary 2005
©
2005 Massachusetts Institute of Technology. All rights reserved.
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Author
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Department of Chemistry
September 17, 2004
,,
Certified by
Harald J. Schwalbe
Professor of Chemistry
Thesis Supervisor
Accepted by
Robert W. Field
Department of Chemistry
Chairman, Departmental Committee on Graduate Students
This doctoral thesis has been examined by a committee of the Department of Chemistry as follows:
•I
Professor Robert G. Griffin
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Chair
Professor Harald J. Schwalbe
Thesis Supervisor
Professor JoAnne Stubbe
...-..
Development of new Parameters for Structure Determination and
Dynamic Investigations on Biomacromolecules by NMR
by
Elke Duchardt
Submitted to the Department of Chemistryin Partial Fulfillment of the Requirementsfor the Degree
of Doctor of Philosophy
ABSTRACT
Nuclear magnetic resonance (NMR) spectroscopy is unique in the content of structural as well
as dynamic information that it can provide at atomic resolution. The aim of this PhD-thesis was to
contribute to the understanding of biochemical processes by means of NMR-spectroscopic
techniques, targeting specific problems as well as contributing to the general understanding and
providing new, widely applicable methods. The main focus was on the structural as well as dynamic
study of ribonucleic acids (RNA).
A new structural NMR method was developed aimed at the determination of the glycosidic
torsion angle X in RNA, which defines the relative orientation of the nucleobases in respect to the
ribose moiety (Duchardt et al., 2004). X was derived from the angle dependence of carbon-hydrogen
dipole-dipole, nitrogen chemical shift anisotropy cross-correlated relaxation rates (F-rates). Method
development comprised the design of a novel NMR experiment, the (HCN), as well as the
parameterization curves. The novel method provides an accuracy of
introduction of F versus
around 10 degrees or better, comparable to the precision of conventional angle determination
techniques. In contrast to conventional methods, the F(HC) is sensitive to molecular size and will
therefore proof beneficial in the investigation of larger RNAs by NMR
Apart from this methodological contribution to RNA structure determination, the dynamic
properties of the abundant YNMG RNA tetraloop motif (with Y=C or U; N= any base; M=C or A)
were studied in a residue specific manner by means of 3C NMR relaxation measurements. The
dynamics of the extraordinarily stable cUUCGg motif were compared to the less stable uCACGg
hairpin, which forms the stem-loop D (SLD) in the regulatory 5'-cloverleaf of coxsackievirus 3B.
Measurements were carried out at 25°C, at which both motifs are stable, as well as at close to the
melting point of SLD (43°().
Ribose and base moiety specific amplitudes and time-scales of motion were extracted from R1,
R1 p and the heteronuclear nOe of Q6and CQ.in pyrimidines and CQand CQ.in purines by application
of the model-free formalism (Lipari and Szabo, 1982). The application of the model-free formalism
to C1., and C6 which possess an additional adjacent carbon spin, was examined for the uniformly
isotope labeled RNA hairpins investigated in this study. In addition, the relaxation data analysis was
optimized for the 13C chemical shift anisotropy based on chemical shift tensors available to date
(Stueber and Grant, 2002;Fiala et al., 2000). While at room temperature, the dynamics closely follow
the structural features of both hairpins, residues in the loop closing as well as in the adjacent basepair exhibit highly increased flexibility at temperatures close to the melting point of SLD. In contrast,
loop dynamics remain unperturbed. In SLD, the residues close to the loop are conserved among the
5
family of rhino- and enteroviruses, indicating a sequence based mechanism of decoupling loop
structure and stability in order to adjust to the twofold requirement of a defined structure for protein
recognition and low stability to ensure efficient melting within the genomic transcription process.
In addition to investigations on RNA, structural studies were also conducted on the C-chain of
the T-cell receptor (TCR) in order to contribute to the elucidation of the early events in T-cell
activation. According to earlier studies (Aivazian and Stem, 2000), the extracellular encounter of the
T-cell receptor with an antigen is transmitted into the T-cell via a lipid induced structural transition
of the TCR C-chaincytoplasmicdomain. In order to lend detailed structural support to the proposed
mechanism, the structure of TCR 4 was studied in free solution and in the presence of detergent
micelles. By means of standard NMR-techniques, the backbone assignment of both forms of 4 was
achieved and secondary structural information was obtained. In addition, the binding site of the
protein Nef of simian immunodeficiencyvirus (SIV) onto TCR ~ was determined.
Thesis Supervisor. Harald Schwalbe
Title:Professor of Chemistry
References:
Aivazian, D. and Stem, L. J. (2000) Nat Sta
Bio, 7, 1023-6.
Duchardt, E., Richter, G, Ohlenschlager,O., Gorlach,M, Wohnert, J. and Schwalbe,H. (2004)J
Am(banSr,
126, 1962-70.
Fiala, R., Czernek, J. and Sklenar, V. (2000) J Bind NMR, 16, 291-302.
Lipari, G. and Szabo, A. (1982) JAm CaenSoc 104, 4546-4559.
Stueber, D. and Grant, D. M. (2002) JAmCIemSc,
124, 10539-51.
6
"Themare a rmillionzs
to get no signal"
Harald Schwalbe
"7n
eejust muke nore scans"
Christian Richter
'"Still,Me iller
gt as rch signl as ona Varian"
Jens W6hnert
7
THANKS TO....
.... THE SUPERVISORS
I am deeply indepted to my thesis supervisor, Prof. Harald Schwalbe. He taught me NMR,
encouraged, supported my projects with his expertise, and especially in the last years of my thesis
supported my increasing independence with occasional insightful discussions.
Jens W6hnert, who would never allow me to call him anything close to my supervisor, has been of
indispensable help to me ever since he joined the group. I would like to acknowledge his critical
support concerning both specific as well as very fundamental aspects of my work his patience in
telling me the same things over and over again (it did go both ways after all) as well as the readiness
in supplying me with any type of literature. I also appreciate his cynical sense of humor. Thanks for
reading my thesis so carefully and partly even twice.
I would like to warmly thank Christian Richter for our inspiring collaboration and his continued
support. It was due to his careful and friendly supervision during a lab-rotation that I decided to
work in the field of NMR Later, collaborating with him in the field of pulse programming proofed
very fruitful for both of us. In addition, his great memory for whatever he had ever done made my
life so much easier, as did his easy manners and his continuous kindness, no matter if a shaped pulse
was required or a ride home.
.... THE THESIS COMMITTEE
A am grateful to Prof. Bob Griffin, Prof. Barbara Imperiali and Prof. Joanne Stubbe for their
support in the course of this thesis, in respect to science as well as in coordinating the whole
procedure.
.... THE JAN GUY
I could not be more grateful for your cheerful temper during this last month of writing. Thanks for
being there, making me worry less, getting me out for a coffee break, feeding me with junk-food and
reading this stuff over and over again. You are the best.
....THE FAMILY
I am deeply grateful to my mother, who always wanted me to go study and become a PhD. I am
even more grateful that in the end she really believed, I would make it. Thank you for starting to
study medicine, so you finally realized that I actually learnt something in the last ten years.
My brother has been always there for me during all this time. He makes me ever so happy to have a
sibling that I always walk around telling people they should have more than just one child.
.... THE SISTERS IN ARMS
Luckily for me, Joanna & Suman both started writing up when I did to share good sessions in the
library, deep depressions about unproductiveness and nice coffee-breaks. Thanks guys, long live the
Tripod!
8
....THE OTHER PHD-STUDENT
For five years, I sat next to Julia, first in Boston then in Frankfurt. That's why we had plenty of time
discussing almost everything from science to an appropriate retirement plan. I cherish your honest
opinions and the occasionalapple that I got from you.
....THE GIRLS-LAB
I had a great time with Julia, Emily, Karla and Aph. Thanks for the special, girly atmosphere. Where
else could I share my waist-line concerns and learn about tanning solutions? My special regards go
to Aph, who became a friend and impressed me both by her dedication to her work and her great
fashion sense. Thanks for your constant open ear for my worries. I also want to acknowledgeKarla,
who is trying so hard to make sense of this thing called thesis at the moment. Don't worry, I did it,
so you can, too.
.... THE GROUP
The continued good accord in the whole Schwalbe group rendered this PhD period a very pleasant
time for me. The aptitude to help other group members, the sense of a common goal and the
extension of the work group into private activities were really exceptional. Among all these people
my special regards go to Christian Schlbrb, who was a very friendly and good humored fellow system
administrator and will certainly do a great job in the future, if he doesn't decide to finish his PhD,
Boris FiIrtig and Jonas Noeske, who were very good undergraduate researchers and fortunately
decided to join the group and Elke Stirnal, who patiently purified my RNA on the HPLC Last but
definitely not least, I would like to thank Frau Paulus, who is simply the best secretary ever and the
gute Geist of the work group. The same goes for Zim, who was always there for helping out with the
spectrometers or anything technical, which is amazing considering his difficult working environment.
.... THE BOSTON PEOPLE
My deep-feklt gratitude goes to my friends in Boston, who helped me to find a start in this new
environment. You contributed a lot to the great experience that these two years abroad constituted
for me, which I still remember in happy melancholy. Especially, I would like to thankJulie, the social
wizard, who introduced me to squash as well as to her very diverse circle of friends and thus gave me
an easy start. In addition, I want to acknowledge Peter and his great explorer spirit. If it wasn't for
him, I would have never seen half as much of New England. He was also a great supporter during
my time at MIT. I would also like to thank the people in the Magnet Lab for their enthusiasm and
their friendliness, especially Ajay and Peter. I feel especially grateful to Chris Turner, who became a
special supporter to me during my time in Boston and is one of the funniest people I have ever met.
9
TABLE OF CONTENTS
Abstract .....................................................................................................................................
5
Acknowledgement ...................................................................................................................
8
Table of Contents .....................................................................................................................
10
List of Abbrev iations................................................................................................................ 13
CHAPTER 1: INTRODUC
ON...........................................................................................15
The power of NMR
16
Basic principles ..............................................................................................................
determination
Structure
Structure
dtermination.................................................................................................1
17
19
Dynamic investigations ..................................................................................................
26
References-30
......................................................................................................................
CHAPTER 2: F(HCN)-EXPERIMENT
............................................................................... 37
INTRODUC17ION
40
I........................................................................................................
The glycosidic torsion angle X
40
Determination of X by NMR
41
Determination of X from cross-correlation relaxation rates
Parameterization of F(X)
Definition of X in NMR structure determination
Molecules under investigation: The 14-nt and the 30-nt RNA
42
44
45
45
MATERIAL AND METHODS..
47
Sample preparation
47
NMR spectroscopy
47
F-rate calculation
RNA structure calculation
49
49
51
............................................................................................................
RE SULTS
The quantitative F(HCN-experiment
The quantitative F(H2'C2'N-experiment
51
68
1
CONCLUSIONS ...........................................................................................................
71
Pulse Sequence Development
72
Experimental Quality
REFERENCES
............................................................................................. 75
10
CHAPTER
3: RNA TETRALOOP DYNAMICS .................................................................. 79
INTRODUCTION
,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,82
The tetraloop RNA secondarystructure motif
Dynamic investigationson RNA by NMR
82
85
Autocorrelated relaxation rates
Model-free formalism
Motional models
88
89
92
MATERIALS
AND
MATERIALS
ANDMETHODS...94
METHODS~~~..................................................................................9
NMR spectroscopy
Relaxation data analysis
RESULTS
94
96
99
.....................
Temperature dependence of the imino proton resonances
99
13C relaxation analysis
101
.
DISCUSSION
........................................................ 131
Model-free analysis
131
Dynamics of the YNMG tetraloop motif
134
OUTOOK .139
,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,140
REFERENCES
CHAPTER 4: STRUCTURE OF THE TCR -CHAIN ........................................................
145
INTRODUCTION.148
~~........................................................................................................4
INTRODUCTION
T-Cell Activation
The TCR ;-chain
TCR q /Nef interaction
Protein backbone resonance assignment
MATERIALS
AND
MATERIALS
ANDMETHODS
METHODS~~~..................................................................................15
148
149
150
152
154
Protein expression
Backbone assignment of free TCR q
Concentration dependent experiments
Temperature dependent experiments
~/Nef interaction studies
TCR q in LMPG micelles
154
154
155
155
155
156
RESULTS
5
.......................................................................
RESULTS
........................................
157
TCR ~ in solution
157
TCR bound to IMPG-micelles
169
CONCLUSIONS ,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,174
REFERENCES
.,,,,,,,,,,,,,,,,,,,,,,,,,,176
11
APPENDIX
...............................................................................................
179
A1. CHAPTER 2: F(HCN)-Experiment ---- ..............................................................
Pulseprograms
F-rate calculation
References
A2. CHAPTER 3: RNA Tetraloop Dynamics. ..............................................................
Pulse sequences
Extraction of peak intensities
181
181
184
195
196
196
202
A3. CHAPTER 4: Structure of the TCR c-chain .......................................................... 203
Assignment of free TCR 4 at 278K
Assignment of free TCR ~ at 317K
Assignment of TCR
tLMPGat 310K
PUBLICATIONS
203
205
207
211
...............................................................................................................
12
LIST OF ABBREVIATIONS
13C
carbon- 13
15N
nitrogen-
1H
hydrogen- 1 (proton)
31p
phosphorous-31
A
adenine
C
cytosine
CD
circulardichroism
cm
centimeter
CS
chemical shift
CSA
chemical shift anisotropy
CSI
chemical shift index
CV
coxsackie virus
DNA
desoxyribonucleic acids
F
cross-correlated relaxation rate
G
Gauss
G
guanine
hetnOe
heteronuclear nuclear Overhauser enhancement
HIV
human immunodeficiencyvirus
HSQC
heteronuclear single quantum correlation spectrum
Hz
Hertz
INEPT
insensitivenucleus enhancement bypolarization transfer
ITAM
immunoreceptor tyrosine-basedactivation motif
J
scalar coupling constant
K
Kelvin
KD
dissoziation constant
kDa
kilodalton
LMPG
myristoyl-phosphatidyl choline
MHC
major histocompatibilitycomplex
15
13
MHz
megahertz
mM
millimolar
PM
micromolar
MQ
multiple quantum
MRI
magnetic resonance imaging
Nef
negative factor
nOe
nuclear Overhauser enhancement
ns
nanoseconds
NS
number of scans
ppm
parts per million
ps
picoseconds
R1
longitudinal relaxation rate
R2
transversal relaxation rate
RDC
residual dipolar coupling
RMSD
root mean square deviation
RNA
ribonucleic acid
S2
square of the generalized order parameter
SIV
simian immunodeficiency virus
SLD
stem-loop D
TPPI
Time Proportional Phase Incrementation
SW
spectral width
Tc
molecular rotational correlation time
TCR
T-cell receptor
TMSP
3-trimethylsilyl propionic-2,2,3,3-d4 acid sodium salt
TOCSY
total correlation spectroscopy
TROSY
transverse relaxation optimized spectroscopy
U
uridine
Amino acids are abbreviated using the common single letter code.
14
Chapter 1:Introduction
CHAPTER
1
Introduction
TABLE OF CONTENTS
1.1
The power of NMR .......................................................................................................................
16
1.2
1.3
Basic principles ................................................................................................................................
Structure Determination...............................................................................................................
17
19
1.3.1
Structure determination process ..........................................................................................
19
1.3.2
Structural Parameters ............................................................................................................
19
1.3.3
RNA structure determination ..............................................................................................
22
1.4
Dynamic Investigations .................................................................................................................
26
1.4.1
The importance of dynamics in the structure-function relationship .
............................
26
1.4.2
Dynamic information from NMR ......................................................................................
28
1.5
References........................................................................................................................................
References301.5
References30
301.5
15
Chapter 1: Introduction
1.1 The power of NMR
Nuclear magnetic resonance (NMR) spectroscopy constitutes a non-invasive technique that allows
for the spatially resolved investigation of objects of the size of a view Angstrom up to the entire
human body (Figure 1). Two fundamentally different techniques have emerged: While the
observation of the same spin in different environments yields super-molecular spatial resolution in
magnetic resonance imaging (MRI) and NMR niCat,
the investigation of different spins in the
In MRI, the spatial distribution as well
same environment constitutes the basis of NMR sptrcpy.
of water hydrogens is obtained within different tissues or cell
as the specific properties
compartments through the application of magnetic field gradients, thus creating highly resolved,
non-invasive two- or three-dimensional images from opaque, water-containing objects such as the
human body. MRI therefore constitutes a powerful medical tool both for research and for diagnostic
purposes. NMR microscopy represents the high spectral resolution variant of NMR, allowing for the
detailed study of objects of the size of a view millimeters down to individual cells. In contrast to
other microscopy techniques, NMR microscopy does not require transparent objects and can be
extended to the observation of specific compounds such as metabolites.
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16
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Chapter 1: Introduction
The major disadvantage of MRI techniques lies in their relative insensitivity, which they share with
all NMR-techniques. Hence, if substances other than the bulk water are to be observed, appropriate
isotope labeling schemes have to be applied, while substances of low in ziw concentrations may not
be detectable at all.
In contrast to MRI techniques, which directly obtain an image of the investigated object from the
spatial distribution of one kind of spins, NMR spectroscopy produces an indirect image of its object
through the observation of different kinds of spins and their interactions. From a network of spinspin interactions, the image of the object is then reconstructed. The observation of different spins
allows for the investigation of objects on the molecular level. Due to the lack of spatial resolution
and sensitivity of this technique, homogenous sample conditions are required. Hence, while it is
possible to conduct these studies under physiological conditions within the living cell [7], sample
inhomogeneities within the cell render this method unfavorable. In addition, NMR spectroscopy is a
rather insensitive technique and therefore requires high sample concentrations. Consequently, the
lion's share of NMR spectroscopic investigations so far have been conducted in zto. To date, the use
of isotope labeling techniques with
13C, 5 N
[8] and 2H [9-11], the development of sensitivity and
resolution enhanced NMR techniques [12-14], the provision of novel structural NMR parameters
[15, 16] and the use of high field magnets have been combined to render the study of proteins of
around 30kDa and oligonucleotidesof around 10kDa a routine task, while moleculesof up to 80kDa
[17] can be studied in high detail in special cases. Due to their unfavorable relaxation behavior,
membrane proteins are still difficult to study in solution state NMR spectroscopy. In contrast, solid
state NMR allows for the study of membrane proteins within the physiologicalenvironment of lipid
bilayers [18, 19]. This technique, however, still suffers from line broadening and low sensitivity
compared to liquid state NMR.
1.2 Basic principles
In the following, a brief introduction to the basic principles of NMR is given. For a general
introduction to NMR, see ref. [20].
NMR experiments are based on the manipulation and detection of weak sample magnetizations
that arises from the interaction of spin-carrying nuclei with a static magnetic field. The simplest
NMR active nuclei are those with a spin of ½. Spin h nuclei can occupy either of two distinct energy
levels or spin-states. In the presence of an external magnetic field, the degeneracy of these spin-states
is removed. The spins will align either parallel (lower energy state) or antiparallel (higher energy state)
17
Chapter 1:Introduction
to the magnetic field. In the classical picture, the spins can be imagined as precessing around an
effector axis oriented along or opposite to the magnetic field. The precession frequency (Larmor-
frequency) is the product of the magnetic field at the position of the spin and a proportionality
constant y, the gyromagnetic ratio, which reflects the magnetic properties of the nucleus. Since the
magnetic field experienced by a given nucleus constitutes the sum of the external magnetic field and
the field generated by the surrounding electrons, resonance frequencies reflect the chemical
composition as well as the conformational arrangement. Under equilibrium conditions, the higher
population of the lower energy spin-state results in a weak excess magnetization oriented along the
magnetic field, which can be manipulated by the application of radio-frequency pulses at a frequency
close to the Larmor-frequency of the nuclei. By application of the appropriate pulses, the
magnetization can be rotated into the plane transversal to the magnetic field, where its precession
frequency can be detected. Due to their different Larmor-frequencies, spins in different chemical
environments are detected at different frequencies, and spectra can be obtained. Since the Larmnor-
frequency or resonance frequency of a spin is dependent on the spectrometer field-strength, the
frequencies are normalized to the magnetic field and then called the chemical shift, which is quoted
in parts per million (ppm).
Perturbation of the equilibrium magnetization also constitutes the starting point for spin-spin
interactions through different through-bond as well as through-space mechanisms (section 1.3.2).
These effects give rise to spectral resolution in multidimensional spectra from the correlation of
different chemical shifts. In addition, spin-spin correlations relate spins in space or through bonds
and therefore constitute the basic principle of any structural NMR spectroscopic study.
Spin
1h2
particles are the ones most widely studied by NMR. Apart from 1 H (protons) and
3 1p,
the
spin ½ isotopes of most elements occurring in macromoleculesare not very abundant. In the case of
carbon and nitrogen, isotope enrichment techniques with 13C (natural abundance 1%) and "5N
(natural abundance 0.4%) have been developed both for proteins [8] and nucleic acids [21-23].
18
Chapter 1: Introduction
1.3 Structure Determination
1.3.1 Structure determination process
Structure determination by NMR is based on the sequence of procedures depicted schematically in
Figure 2. At first, a sample of the molecule of interest needs to be prepared in quantities of several
milligrams featuring the desired isotope labeling scheme. In general, several samples with different
labeling schemes are required. After preparation, the desired NMR spectra are obtained from the
samples. Spectra analysis requires the resonance assignment in order to connect the resonances
within the spectra to the spins in the molecule. The NMR parameters, on which the structural
investigation is based, are then obtained from the assigned spectra and subjected to a structure
calculation procedure.
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1.3.2 Structural Parameters
The acquisition of structural parameters from NMR-experiments is possible due to distance or
angle dependent spin-spin interactions. The most common structural parameters are summarized in
Figure 3 and briefly discussed in the following:
In general, distance information is obtained exclusively from dipolar through space interactions
(nuclear Overhauser effect or nOe [24). The intensity of a nOe between two nuclei is dependent on
the inverse sixth power of the internuclear distance. Proton-proton nOes constitute the main source
of information in NMR structure determination [25]. They, however, only report on distances
smaller than 6 A and therefore fail to report on global structural arrangements.
19
Chapter 1:Introduction
TRADITIONAL TECHNIQUES
NEW TECHNIQUES
I
a
I
Figure 3: StructuralNMR paranreters
and dxheir
inpliatin instmcturedetemina
The same applies to scalar coupling constants (J-couplings) [26]. J-couplings are angle dependent
on through-bond interactions. While three-bond vicinal J-couplings (3J-couplings), which report on
dihedral angles, represent the most widely used structural parameters [27], also geminal (2J)and onebond (J) couplings can contain a measure of structural information [28-30]. The structural content
of scalar couplings cannot be extracted directly but relies on semi-empirical parameterizations, the
so-called Karplus-equations [31]. For a number of structurally interesting J-couplings, e. g. for 3J(HNH-) in the protein backbone, these Karplus relations have been developed extensively[32-35].
Cross-correlated relaxation rates (F-rates) have only recently been introduced as structural NMR
parameters [15] and since their introduction have found wide-spread application in structural studies
on proteins and as well as nucleic acids [12, 36-41].
F-rates arise from the angle dependent through space interaction of two dipoles (Figure 3), a dipole
and the chemical shift anisotropy (CSA) of a nucleus or two CSAs. Although the chemical shift
anisotropy is averaged out in solution by rapid tumbling and only the isotropic chemical shift is
observed, the CSA results in relaxation relevant fluctuating magnetic fields at the position of
neighboring spin upon molecular reorientation. In principle, the information content of F-rates and
20
Chapter 1: Introduction
J-couplings is redundant. Since F-rates are, however, through space in nature, they do not depend on
through bond correlations and are in principal distance independent. Furthermore, while scalar
coupling constants are independent of molecular size, F-rates are sensitive to the size and shape of
the molecule as well as its dynamics. While these dependencies can complicate the structural
interpretation of F-rates, in the study of larger systems their size dependence constitutes a major
advantage compared to J-couplings.
Residual dipolar couplings (RDCs) represent the most easily accessible and therefore most widely
used long-range structural NMR parameter. RDCs report on the orientation of two dipoles in
respect to a molecular orientation tensor (Figure 3) [16, 42, 43]. The dipolar interaction between two
nuclei depends on the angle between the internuclear vector and the magnetic field and can be of the
order of several Kilohertz. In isotropic solution, dipolar interactions are averaged out due to rapid
molecular reorientation and are only maintained in the nOe and their contribution to nuclear
relaxation. Weak alignment by orienting media such as phage or lipid bilayers leads to the adoption
of a preferential orientation of a fraction of the molecules, thus rendering the measurement of scaled
or 'residual' dipolar couplings possible. These RDCs are of the order of several Hertz and can be
translated into structural information through a molecular alignment tensor, which defines the extent
of alignment and the orientation of the molecule with respect to the magnetic field. Since RDCs
describe the orientation of each dipole in respect to this alignment tensor, relative orientations
between different dipoles can be obtained irrespective of the distance. Similar information to the
ones obtained from RDCs can also be extracted in the same fashion from the CSA [44-46].
Apart from these spin-spin interactions, the chemical shift of a spin by itself is sensitive to its
environment and therefore carries structural information. In proteins FL, Cu, Co and C chemical
shift deviations from standard random coil chemical shifts measured semi-quantitatively in the form
of the so-called chemical shift index (CSI) constitute a very sensitive probe for the presence of ahelical of 13-sheet secondary structural elements [47-49]. In addition, chemical shift changes
constitute sensitive probes for structural rearrangements involved in conformational transitions or
interactions.
In this thesis, a structural investigation of the C-chain of the T-cell receptor has been carried out
restricted to standard protein assignment experiments in order to obtain chemical shift based
secondary structure information as well as to study the interaction site with the HIV-protein Nef
(Chapter 4).
21
Chapter 1: Introduction
1.3.3 RNA structure determination
Ribonucleic acids (RNAs) are composed of a sequence of four different nucleotides, adenosine
(A), cytosine (C), guanosine (G) and uridine () (Figure 4A), linked by phosphodiester bonds. The
ability of the nucleobases to form hydrogen bonded pairs constitutes the basis of secondary structure
formation. While regions with Watson-Crick base pairs (A paired to U, C paired to G; Figure 4B)
fold into helical duplex structures, loops are formed when the RNA strand folds back on itself and
bulges arise from unpaired regions within the stem (Figure 4).
These basic secondary structure
elements are structured to a varying degree, involving non-canonical (non-Watson-Crick) basepairing as well as hydrogen bonding interactions of the ribose moiety and the phosphodiesterbackbone.
",~i'
~~
-.NH
2
A
B
- ... ..
P' 0
, .:.:
-
.... .
: .I.' ..
'.'
''-..
, ':'.,:
..
..... ::":-
..,:'
..
.
.. .
,:.!):.i'
" -. :i.:.
Nucleo-
N
abase
:(IA]
:
:'
J
N
III
i
0~
Phosphate
IRibose
IH Im.i.JI
OH
"H
OH
=
i
.--.
r . ':...
...
.
..
NH
2
'".
:::.-:
---
'
.
...
NI
I
I
I
. 1: , ; - I . I I . ., ,, ,. .
".:"":
f-:"
NH
N'
:'.
'
u
NH2
I
. II I II
Z. .
:
I
Z
.
I
.
.I , .
.
.
. II. I .,
,:
: - --.
i.
,:
...I I %5 1....%, I
.
....Z
'
. III
C
-.::--/..-~ ... ' .- "-:..-'--.... "
Duplex
, ..
0--
.0
.....
.. '- ..::..- :...-..":......:~.::!: ...
Loop
--
Base
Base-pai
.. :..:.. : '.- !.-'':':
Bulge
''Three-way
'
-I-I ;
II
Figure 4:RNA srumo A Nudetde sm
.:..
'
.:..''.:..'..
'=.- ;''
~.:"i' :'."'..l',
Four-way
IJunction Junction
|
=
.--
.:.-:';..".:..
' G-Quartett ',
Guanine.
i
2'I~~~~~~~~~~~~~~~~~7
J~l 00S_10
J=QI0l
7?
B: WatscnCride
basepas. C Semdarystru
senivysctim nag (nit).
andnve owplwaiud
22
h
boxks (left)
Chapter 1: Introduction
Apart from a small number of special secondary structure motifs, like the G-quartett, more
complicated secondary structure arrangements such as three- or four-way-junctions (Figure 4C) can
be derived from these three basic secondary structure motifs.
Since the investigation of RNA by multidimensional heteronuclear NMR methods was made
3C
possible due to the development of techniques for the preparation of
and "5 N isotope labeled
RNA in 1992 [21-23, 50], a large number of NMR experiments has been devised [51, 52] aimed at
improving the accuracy of NMR-derived RNA structures. However, the size-limit of RNAs studied
routinely by NMR is still restricted to around 10kDa (- 30nt). This size limitation is due to the
inherent low spectral resolution of oligonucleotides. As depicted in Figure 5A for a 10-nt RNA with
3C
selective
labeling of the four loop residues [53, 54], the poor spectral resolution of proton 1D
spectra can be overcome by 2-dimensional correlations with the attached carbon nuclei.
A
B
]IFtnhII
IJ 11
'-U
|
..
U.'
.
;,..-
Cl,
U
u Cc
.-.'
-.U
. . .' - .. ..
C-G
..
,-
G!'
.
. . '-
..-''
i' .;:.'HS";:'.:
u^Jf .-..ui. !.
....
... .:.:i.i
.
.-'.:---U -'
., .;.'-.. .. . :
N..... .;. ~~~~~~~
.";".-i:
..
/
~~~~~~~
... . ....
. °
- * : 1..C....
t-
.......
6.0 .5
- - :
.
5
:
G
'
.'
.45
-.';.:..'.,
....
...
C
.
..
.:
..
. ;-..:.
.........
-sc
....
..
.
~~~~~~~~~65-
:
....
1H-- -
4
s , .....,.,,..,..
~C5'/H5',H5"
---
:
..
a.
. '.... ... :....
.... -.. ....
...
C2'/H2'
C3'/H3'...
-'=75....
..7 .: '
.
'.~ '.,':';,-1:
.-C
'.: :G
' '-:.:
:1,'J .'.'501.':..
G-G
6a :-.' '.'.
55
-. : :.
C4!IH4'
::-C'/H1'..:
'
"
.......
-:....
..
... . . ...
.:.0 5 .:5,0'4.4
~~~~~~~~~~
Figure 5: 1Dprnm spenn ad
(t)
13Claktbeling
in thef
.....
':'--3:'.'.:
".
-'.'!.:".:
"..
"..
....
:.
-AC
-G
UA
::-G-C
-
;
;:-..
4.5-
U" :'
.
.....
.
.....
" .:....;
'.... .s.
... ...
.
.
-
4:-.-': :.
4,5
.......
:.^.,^.,.....
.--,.u;*
'H,
5~~~~~ - :.
..
C:"...
............
.A
85:'; ,'
-1-4.
,-!
.-.
:92 .'. "
-:
4
1CI
5'
(t
qfd'
e risberamties f a 10-nthapn
lct residues
(). 'H, 13C-ordati (H, C- rgi
of afidly '13Clali
RNA haitpin The lo ard dosingbasepairresiduesarehigblild in de speanan
23
.. ..'..::..'
3'
ithsdect
14-nt (B) ard 30-nt (C)
Chapter1: Introduction
In contrast, for larger molecules, like a fully 13Glabeled 14-nt (Figure 5B) and a 30-nt hairpin RNA
(Figure 5QC,the spectral resolution becomes increasingly poor. Dispersion is obtained mainly for the
non-canonical loop regions, which exhibit structure specific resonance patterns (see grey shaded
resonances in Figure 5B,C), while the resolution of canonical duplex structures resonances is small.
Spectral overlap arises from the small number of building blocks of partly similar or even identical
chemical constitution as well as the limited diversity in secondary structural motifs. Although spectral
crowding can be overcome by the application of elaborate selective isotope labeling schemes [53],
this is work as well as cost intensive.
A
B
1
... .: .. ,.
, .. '. :' "'., ., .
C
. ..
l
I . ...
,
:
,: o
Figure 6: Tosionangl (4) andNMR-atew mndez
(B) in dtephapt
ter dei
ck and e riboie ty ofRNA. C
Basesin RNA. The mdedzsf arepai
it tir
pesiz WatsmoCrideKdren b
g panrs; amiei (A) it
uradle (U) and guarni (G)
amaddioUy mk edby ba
ithcyeid (C). Pnta
are shded in gre prom, ubich armecang
str .
24
uith thesdolet O er
Chapter 1: Introduction
In addition to the problem of spectral resolution, the density of structural NMR parameters in
RNA is small compared to its degrees of freedom In the absence of conformational averaging, the
structure of a single oligonucleotide can be described by six dihedral angles (-c)
in the backbone,
two parameters for the cyclic ribose (the pseudorotation phase P and the maximum amplitude v-)
and the glycosidictorsion angle X, which defines the orientation of the nucleobase in respect to the
ribose (Figure 6A). Due to the depletion in NMR-active nuclei (Figure 6B), the backbone
conformation cannot be sufficiently described by conventional NMR parameters such as 'H-H
nOes and lHI,1H-,
13G
and 1H,3 1P-coupling constants. In particular, the angles a and ( cannot be
determined. Only recently, a method based on GH
dipole-dipole,
31 P-CSA
cross-correlated
relaxation has been developed to address a and 4 [55].
In contrast, the ribose conformation can be determined from homonuclear 3J(H,)
coupling
constants [56-58] or GH dipole-dipole, C-H dipole-dipole cross-correlated relaxation rates [59, 60].
In the case of conformational averaging, heteronuclear
3 J(CH)
and 2J(C,)
coupling constants [2,
58, 61, 62] can complement the structural characterization of the ribose.
The glycosidic torsion angle X, is conventionally determined from nOe intensities between base
and ribose protons [63] or 3J(C,)
coupling constants [64-66]. The advantages of a new method
based on GH dipole-dipole, N CSA cross-correlated relaxation rates [1] developed in the course of
this thesis, are discussed in Chapter 2. Within the planar nucleobases, the number of protons is also
scarce (Figure 6).
In addition, the solvent exchangeable amino and iino
protons cannot be
detected unless stabilized by hydrogen bonding. The participation of a base imino proton in a
hydrogen bond is hence demonstrated by the appearance of its resonance in the NMR-spectrumr In
addition, the hydrogen bonding partner can be characterized by exploitation of the partial throughbond nature of hydrogen bonds, which gives rise to a scalar coupling between the donor and the
acceptor nucleus [67]. As described in detail above, further interresidual structural parameters can be
obtained from homonuclear nOes and RDCs.
25
Chapter 1: Introduction
1.4 Dynamic Investigations
1.4.1 The importance of dynamics in the structure-function relationship
In the enterprise of understanding physiological processes, it is in general easier to find out
bat
something is doing than to elucidate how it exerts its function. In order to understand molecular
function, detailed structural knowledge is indispensable. It is, however, a common feature of all
structure elucidation techniques like NMR, X-ray or electron microscopy that they report only on
either the averaged structure or the most populated structure, depending on the time-scale of
However, due to their intrinsic flexibility, molecules do not exist in one
interconversion.
conformation alone but in an ensemble of structures, which is characterized by the populations of
the respective members as well as the rates of interconversion. While in some cases the limited
information obtained from an averaged structure proofs sufficient to understand a mechanism
(Figure 7A), in others the average structure fails to explain the function (Figure 7B).
Averaged Structure
Function
Structural Ensemble
.......
. .....
- .-. -.
..
Averaged Structure
Structural Ensemble
Function
...............
'' -,':,'',',!''': .... - ,-,
. -: - a'-- -,
"
:
B
~~~~b.
f
:
..-
<::
/:
-_
:-
,,
.
.
'..
.......
- '
:
1.
.:.:,
.
,
:.: '
,
.-
:
',
'.,
' '.'
.: ..--.
: .:--
'
.
§. 'a',
'
< '
..
,,, .':
-
''
'
,
.
. ''
'" /
, -Ai
;
''
.,
.
-:
-' .,. '- .'^' ''.
.
7,
.;
-,
:,
:
,
'.
, .'- :.,:
.........
.
:.
1,
.,,:
'1:
' .:"
.
':
:
.;
.
'
'.
.. ,:
ss
............................
,' _JJj,,_','.
'
d','
-,'M§,-',x'''.
! ;;;
.
A- - hi:, * ., ., a.,,.,,-.,2.-, ., ,...:, . s .
.. -':
*
.'.
.............
-
:
' . ' ,' ': .
i,
.
I'd:'.
44A
-''; ,:
''''.'' ".,''''''k''
^ '.,
: A-.
. :-,'-!
' ' .':'
: j
.. f....:
.
.
.. -
'
.
...
,,: .
.-'
''-.'-''-... ., '-.1' , - .' . '
-- 9'; -- -;'' ', ' '';:
',
', -. ', ,: '^
.:"..'
,'.
:.
Figur 7 S tw-Fai
.)...:,:.-.. :.- ::
':. S
,
- --- :
.- .. ^' ., .: . :',, -.'.:.
Thou',:...
...
.
-
- -.
:,
- -- :.
,
rdatiomhp
(4 andStuaural
EmenbleF
wtkn ndatimhp
(B).
26
Chapter 1: Introduction
A very prominent example for the break-down of the simplifying structure-function relationship
can be found in the catalysis mechanism of peptide bond formation in the ribosome. Despite the
availability of high resolution crystal structures [68, 69], the exact mechanism of catalysis is still highly
under debate [68-73]. Also for a number of enzymatically active RNA molecules (the hammerhead
ribozyme [74-76] and the lead dependent enzyme [77, 78), the averaged structure alone was
insufficient to understand the proposed catalyticmechanisms. In these cases, the active form of the
molecule, in which it exerts its function, constitutes a state within the structural ensemble, which is
not observed in the structural investigation.
In order to detect this active conformation, the equilibrium within an ensemble can be perturbed in
a way that the active species become directly observable (Figure 8). An example for this strategy is
the trapping of enzyme transition states by binding to substrate analogs mimicking the transition
state. Apart from that, a possibility of assessing the entire conformational space accessible to a
molecule is to study its dynamics (Figure 8). Dynamic investigations can be carried out without
perturbation of the system and provide indirect information about the structural ensemble through
amplitudes and frequencies of deviations from the averaged or most populated structure. Therefore,
dynamic information
constitutes the key for deconvoluting of the structural average into an
ensemble of states (Figure 7B).
Dynamics
Trapping
:'''-.'
1.
''
-- ;-,.
'
.........
i. -.- '. ,: - - . - --/ .................
--!5;.."-;
--.'--
i
i
.'
:
A
f-.Ad
d
.
;
,,
,
.
,
.
.
- S .
.
.
.
.-
J
Figure 8:Medxs fr
st
om f
emenii.er.
For proteins, the importance of non-equilibrium conformations for exertion of function has been
derived from the correlation of enzymatic activity and conformational flexibility in enzymes from
thermophilic and mesophilic organisms [79, 80]. While these homologues exhibit widely different
temperature optima, internal dynamics are similar at maximum activity. The requirement of a certain
measure of flexibility for enzyme activity was assessed from the dynamic information obtained by
27
Chapter1: Introduction
various techniques such as neutron scattering [81-83], NMR hydrogen-deuterium exchange [80, 81,
84-90], NMR relaxation measurements [79, 91] and molecular dynamics simulations [92-95].
For RNA, the binding mode of TAR RNA to the Tat protein in HIV could be identified as a
conformational capture rather than an induced-fitmechanism by demonstratingthat the bound form
of the RNA already constitutes a part of conformational ensemble of the free molecule [96]. In this
case, the structural ensemble was studied by the dynamic information included in residual dipolar
couplings. This finding indicates that ground state flexibility might constitute the key precursor of
adaptive binding, which is ubiquitously observed for RNA-protein interactions, and demonstrates
the importance of dynamic investigations for the understanding of fundamental biochemical
processes.
1.4.2
Dynamic information from NMR
NMR-spectroscopy is sensitive to motions within a wide range of time-scales (Figure 9). In
principle, every structural parameter also contains dynamic information due to its inherent sensitivity
to structural changes. Apart from these side-effects of mainly structural parameters, a number of
purely dynamic techniques have been developed.
,r
I
...
Z"
I-
1012
10-9
1o-,
10-3
10 °
103
Ai;i
Figure 9: Timsades qfndear nar
28
andNMR-MJ
e.
Chapter 1: Introduction
Different techniques exhibit sensitivity to different motional time-regimrnes.Thus, hydrogendeuterium exchange rates report on the dynamics of solvent-exchangeable protons such as amideand imino-protons on the time-scale of seconds to days. Autocorrelated spin relaxation rates (R1, R2
and the hetnOe) are sensitive to motions in the picosecond up to nanosecond range. In addition, R2
rates as well as ZZ-exchange
spectroscopy
contain information
on slower dynamics in the
millisecond regime. Furthermore, scalar couplings, cross-correlated relaxation rates, the nOe and
residual dipolar couplings are all sensitive to motions in the millisecond regime.
Due to their sensitivity to a wide range of time-scales, autocorrelated relaxation rates constitute a
valuable tool for dynamic investigations,especiallyfor the study of fast internal motions. In contrast
to dipolar couplings, they can be measured without perturbation of the sample with aligning media.
In this thesis, autocorrelated relaxation parameters have been obtained for an abundant RNA
secondary structure motif and translated into time-scales and amplitudes of motion in order to gain
insight into the motifs function.
29
Chapter 1: Introduction
1.5 References
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2.
Duchardt, E.; Richter, C; Reif, B.; Glaser, S. J.; Engels, J. W.; Griesinger, C; Schwalbe, H.
(2001). Measurement of 2 J(HC)- and 3 J(HQC)-coupling constants by alpha/beta selective
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3.
Duchardt, E. and Schwalbe, H. (2004). Residue specific dynamic investigation of the
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4.
Albert, M.; Seebach, D.; Duchardt, E.; Schwalbe, H (2002). Synthesis and NMR Analysis in
Solution of Oligo(3-hydroyAmnoic
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5.
ta, 85, 633-658.
Gee, P. J.; Hamprecht, F. A.; Schuler, L. D.; van Gunsteren, W. F.; Duchardt, E.; Schwalbe,
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Chapter 1: Introduction
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Carlomagno, T.; Blommers, M. J.; Meiler, J.; Cuenoud, B.; Griesinger, C (2001).
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application to an extraordinarily stable 2'-aminoethox3ymodified oligonucleotide triplex. JAm
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Chapter 1: Introduction
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Chapter 1: Introduction
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Chapter1: Introduction
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36
He
..
Chapter2: F(HCN)-Experiment
CHAPTER 2
Determination of the glycosidic torsion angle X in
oligonucleotidesfrom GH dipole-dipole,N CSA
cross-correlated relaxation rates
37
Chapter2: F(HCN)-Experiment
Specific aims
The aim of this part of my research was to introduce a new method for the determination of the
glycosidic torsion angle %in oligonucleotides based on GH dipole-dipole, N CSA cross-correlated
relaxation rates
ric
'
c s A
D
(cNs
). Three slightly different methods were investigated, all relying on
.For the first time, F-rates were introduced as a ayt
use structural parameter. A number
of simplifying assumptions were applied and investigated in respect to their effect on the accuracy of
the technique.
In order to evaluate the new methods, they have been applied to two RNA molecules of similar
secondary structure but different size (14-nt and 30-nt). Investigation of the same structural motif
within two molecules of different size rendered it possible to assess
(i)
the quality of the underlying parameterization and the validity of the simplifying
assumptions, and
the sensitivity and possible molecular size limitations of the method
(i)
,both in comparison to each other and to the currently available torsion angle determination
method from 3 J-couplingconstants. In order to test the relevance of the new parameter, it has been
incorporated into a structure calculation process.
TABLE OF CONTENTS
2.1
INTRODUCTION ..................................................................................................
40
40
2.1.1
The glycosidic torsion angle X............................................................................................
2.1.2
2.1.3
41
Determination of X byNMR ..............................................................................................
42
.
.........................................
Determination of X from cross-correlation relaxation rates
2.1.3.1
Determination of Xfrom rFC' cs
1 1 9
.
................................................................
42
.43
rCocs4
. . .............
DcsA9
and "C6/868,N1/9.43
2.1.3.2
Determination of Xfrom the ratio of r C1'HI',Nl9
A
........................................................................ 43
2.1.3.3 Determination of X;from r' o ~CD
2.1.3.3 Determinationof Xfrom pDDTCSA
4
...
44
...................................................................................................
2.1.4
Parameterizationof r(X).
.
2.1.5
Definition of X in NMR structure determination
38
.......................................
45
Chapter 2: F(HC-Experiment
2.1.6
2.2
Molecules under investigation: The 14-nt and the 30-nt RNA .
....................................
45
METHODS AND MATERIAL ............................................................................... 47
2.2.1
2.2.2
Sample preparation ...............................................................................................................
47
N MR spectroscopy ..............................................................................................................
47
2.2.2.1
F(HCN)-experiment
........................................................................................................
47
3J(C,H)
2.2.2.2
coupling constants ..............................................................................................
48
2.2.2.3
Autocorrelated relaxation rates ......................................................................................
48
2.2.3
F-rate calculation ..................................................................................................................
49
2.2.4
RNA structure calculation ...................................................................................................
49
2.3
RESULTS .................................................................................................................
51
2.3.1
The quantitative F(HC -experiment ...............................................................................
51
2.3.1.1
Pulse sequence ..................................................................................................................
51
2.3.1.2
Derivation of F(X) parameterizations ...........................................................................
55
2.3.1.3
Determination of Xfrom rFHD,CSj ...........................................................................
59
2.3.1.4
2.3.1.5
2.3.1.6
2.3.1.7
Determination of X from F-ratio....................................................................................
61
15
The N CS-tensor in solution from F-base..................................................................63
Transfer to larger systems ...............................................................................................
65
Incorporation of rF,,CSA
into RNA structure calculation .................................66
2.3.2
The quantitative F(H2'C2N-experiment ........................................................................
68
2.3.2.1
Pulse sequence ..................................................................................................................
68
2.3.2.2
Resolving X angle ambiguities with r 2DDCAI 9 .........................................................69
2.4
2.4.1
2.4.2
2.5
CONCLUSIONS
......................................................................................................
71
Pulse Sequence Developm ent .............................................................................................
71
Experim ental Quality ...........................................................................................................
72
REFERENCES ........................................................................................................
39
75
Chapter 2: F(HCN)-Experiment
2.1 INTRODUCTION
2.1.1 The glycosidic torsion angle X
Purines
Pyrimidines
X
27
Figwr 1:D az ofantiandf5 ,fm
pyinid (nit) lia inRNA amgwnfordz
7hepiaur hIs kbenmr4
1 raw f X (nid). Stel lyprd
o5mun i di
pptu wanw rihe
mg forpum (I) and
leaidz,C2-andCye rla
fumEr/[1l].
The torsion angle X in oligonucleotides is defined as the dihedral angle between C4 , N9 , C1 . and 04
in purines and C2 , N1 , C1, and 04 in pyrimiidines. It describes the orientation of the nucleobase with
respect to the ribose moiety. The nomenclature for X is given in Figure 1 [ 1].
The sterically possible X angles in nucleotides are base-type and ribose pucker specific (Figure 1).
Figure 2 shows the
angle distribution for the different nucleotides within the large ribosomal
subunit [2].
I
jnos
ziJI I
-1
7n/)
_/I.I!i,
I
I
0
90
!
180
r
270
270
360
360
x [oN
Figure2: argledisbution in thelaresuboutoftheriha e[2].
40
Chapter 2: F(HCN)-Experiment
In general, the angle distribution is more disperse for purines than for pyrimidines.Not only is the
distribution broader in the ant region, which is the conformation in canonical stem regions, but
purines are also more often found in the non-canonical syn conformation.
2.1.2 Determination of Xby NMR
By NMR, the glycosidictorsion angle can be determined from the distance information contained
in the intensity of the H 1-H 6 (pyrimidines) or H1 -H 8 (purines) cross peaks (Figure 3) in a NOESY
spectrum with an accuracy of about + 50 ° [3], resulting in a qualitative discrimination between the
syn and the ami conformation.
NH9
N
nOe
J(C,H)
OH
Figure 3: CGulid
ndn
fiane of adne Forpyriidir,
OH
fr * anle detemntin by NMR. he irteraig nudeiare inat
z
dn
e
nt maei in ehse are H6forhe
xnoe andC6ad C4
for
in the kdar
e3(C,H) wplirg
Ct7stM5.
A more accurate determination of X can be achieved from a more detailed nOe analysis including
the H2-H61 8 and H3-H6 /8 nOes [3]. However, this analysis is very time consuming and not very
precise due to various effects such as spin diffusion. Alternatively,
can be derived from the
interpretation of 3 J(C2/4 ,H) and 3J(C6 / 8,H1 ,) coupling constants (Figure 3) [4-6]. Recently, refined
Karplus equations have been provided for
3 J(C,H
1 ),
3 J(C ,H ), 3 J(q,H.)
4 1
and
3 J(C,Hl)
for the
different nucleotides of DNA [6]. However, the validity of these equations for RNA still remains to
be investigated. In addition, coupling constant determination is prone to systematic errors for larger
molecules [4, 5].
41
Chapter2: F(HCN)-Experiment
2.1.3 Detenrmination of Xfrom cross-correlation relaxation rates
2.1.3.1
Detenrmination of
from
Cl,1CS,9
The cross-correlated relaxation rate (F-rate) between the q,.-H 1 . dipole-dipole vector and the
chemical shift anisotropy (CSA) of N1 in pyrimidines or N9 in purines,
FCDm
I
,
9
(Figre 4A), is
sensitive to the glycosidic torsion angle.
(TN
33
OH
OH
CY1
A
Figum 4: Sdxzi
nomr
N2 U22C
'-22
B
cF
rentati
DDcsA
(4) and ,
DDCSA
OH
OH
a
I
(inaden
C
B.
orad an
nawe~id~CHI',NI/9
In the simple case of isotropic molecular motion and isotropic fast time-scale internal motion, GH
dipole-dipole, N CSA cross-correlated relaxation rates are given by Eq. (1) [7]:
rF D,CSA = _
P
(1)
HN
154;
o rNYr
Y hS 2
T,
r(Ho,
*[cl (3cos2 c
-1 ) + 42
(3cs
2 02
e
-1)+
33(3cos
33
)]
In this, u is the permeability of the vacuum; h is Planck's number divided by 27; O(Nis the nitrogen
Larmor frequency, yi is the magnetogyric ratio of the nucleus i; rH is the length of the CH dipoledipole vector,
is the rotational correlation time of the molecule; S2 is the generalized order
parameter, cri is the ith component of the fully anisotropic nitrogen chemical shift tensor
(CS-
tensor) and 0ic H ' N are the projection angles between the C-H dipole-dipole vector and the it
42
Chapter 2: F(HCN)-Experiment
' are
component of the nitrogen CS-tensor. In the case of cm
r nteCIH1',NI9
DD,csa
the projection angles OiCHN
dependent on X (Figure 4B).
Eq. (1) shows that the angular interpretation of
rF7'
9
depends
s
on the knowledge of the 15N
CS-tensor of N1 and N9 in general as well as the individual motional properties of the molecule
represented by S2 and -. In order to acquire S2 and
c,
separate autocorrelated relaxation rate
measurements have to be carried out for each molecule under investigation.
2.1.3.2 Determination of Xfrom the ratio of rCCI'',N1/9
DDCSA
and rcDCsA
8H6/8,N/9
Determination
of
addition to FCoIN/
becomes independent of
9
.
c, if rC6D1'N19
(Figure 4
C8H6/,19Fiue4)iacurdn
is acquired in
In contrast to the latter, the F-rate between the C-H dipole-dipole vector in
the nucleobase adjacent to the glycosidiclinkage (C6H6 or CgH) and the CSA of the nitrogen in the
glycosidic linkage (N1 or N)
D,~CSA
I/ 9
'
rC,'HIN
19
andDS
and 'c68H68,
6
NI/9
reports on a conformationally
rigid unit. The ratio between
(F-rtatio)is givenby Eq. (2):
S2
DDcsA
(2) rCHP.N
C 'HI'.N
f, DD,CSA
C6/H6/ 8 ,N
2
C6/8H6/8N
* Cr
(3cos 20 C'H',N
-1) + a (3COS2Ci'H
(3cos1)+UN
20CrH',N _I)
Orl {DO
11
'-l)+'22
"22
033
33$
o (3cos 2 C H6/8,N - 1)+
N (3cos 2 9 C6/8H6/8,N _1)+ aN (3cos 2 C6/8H6/8,N _1)
Vl I1
22
"22
'
33
v33
Under the assumption that the relative motion of the CH dipoles in the ribose and the base
moiety can be neglected and therefore S1HN
ScrmH,,Nandan
C6/8H6/8,Nar
are similar, the F-ratio is only
dependent on X and the characteristicsof the nitrogen CS-tensors.
2.1.3.3 Determination of
from rC'
o CSA 9
"C2'Hl',N19
As for 3J coupling constants the angle dependence of F is degenerate. In order to remove this
degeneracy, additional
CD'
2tcD2vH,
N/
9
dependent
parameters have to be obtained. The determination of
Figure
5)
(Figure
5) provides
provides an
an additional
additional X sensitive
sensitive parameter
parameter and
and is
is methodologicaysimar
methodologically similar to
to
43
Chapter2: F(HCN)-Experiment
the measurement of
rF ' 1,
1 9
However, due to the additional dependence of this F-rate on the
ribose pucker mode, it can only be applied in cases where the ribose conformation is characterized
and rigid.
NH,
rtae otf
Figure5:S·eic
2, NI/9
2.1.4 Parameterization of F(X)
In order to translate the measured F-rates into
values, F(X) parameterizations need to be
established. The level of detail involved in the interpretation of F-rates depends on the extent of
structural and dynamic molecular information included in the analysis. In contrast to coupling
constants, F-rates depend on the apparent size of the investigated moiety, signifyingthat they are
dependent on the size and the shape of the molecule as well as its internal dynamics. Furthermore,
" DDCSA
HCN
-rates depend on information about the
purines. For oligonucleotides in solution, no
N -tensors of N in pyrimindines and N in
1 5N CS-tensors
have been determined so far, since the
chemical shift is averaged due to rapid tumbling and the measurement of a fully anisotropic tensor
therefore requires elaborate experimental schemes. However, nitrogen CS-tensor values and
orientations reported for mononucleoside crystals determined by solid state NMR [8] can be used in
the parameterizationof
rFCSA
. In
order to introduce F-rates as n
touse structural parameters in
the same way as coupling constants, it was assumed that (i) 15N CS-tensors obtained for
mononucleosides in the solid state can be applied to oligonucleotides in solution and effects
stemming from (ii) anisotropic diffusion, () internal motion (S2 =1), and, in the case of the F-ratio,
(iv) differential motion between the base and the ribose moiety can be neglected.
44
Chapter2: F(HCN)-Experiment
2.1.5 Definition of X in NMR structure determination
In conventional RNA structure determination by NMR the glycosidic bond angle X is restricted to
either syn or anti from the analysis of the HI-H6/ 8 nOe. X angle restraints determined from the
measurement of F-rates offer a way to incorporate more direct torsion angle information into the
structure calculation. In addition, while extraction of the F-rates constitutes a linear task, in the case
of nOe information, both of the involved protons have to be identified, thus increasing the
complexityof the process.
2.1.6 Molecules under investigation: The 14-ntand the 30-nt RNA
The 14-nt RNA (Figure 6A) was selected as a model compound to evaluate the accuracy of X angle
determination from /-'DocSA since it is conformationally rigid and contains both canonical stem
regions with a low divergence in X and a non-canonical cUUCGg tetraloop, exhibiting a broader
distribution of
angles. In addition, the tetraloop comprises an unusual U:Gsn, while all other
residues adopt the ami conformation.
A
B
U7 C8
U6
C16
A C 16
C 14 ----------G
----- G9
I0
C5-G
A 4 - U11
U ? -- G18
A12 U9
C 3 -G12
U11-
A2g
G10
C2 1
G2 C13
GI- C
m
G1 C14
5'
9
cG
- C 22
3 4
3'
-
G26
A-
C3 _ G 2 8
G 2 - U*;
G 1 - C30
5'
Figure 6: Priny
shoe inbx,
satine and seorndarys uaum ef the 14-nt (
unaM
oEs bigmy
45
3'
and dx 30-nt RNA (B).
13
Q
N lded
eske am
Chapter2: F(HCN)-Experiment
Autocorrelated relaxation rates demonstrate that all residues but for the base of U7 in the loop are
rigid at the temperature of 298K, at which the experiments have been conducted (see Chapter 3).
Homonuclear
3J
coupling constants indicate that the ribose moieties of all residues are fixed in one
conformation. Although there is no structure available of the 14-nt RNA to date, the structure of
cUUCGg-tetraloops has been consistently characterized by both NMR [9] and X-ray crystallography
[10]. The 30-nt RNA (Figure 6B; PDB entry 1RFR) consists of two canonical A-form stem regions
connected by three pyrinidine mismatch base pairs (U:U, QU, U:U) and an uCACGg tetraloop,
which has been shown by NMR [11] to adopt a structure similar to the one of the cUUCGg
tetraloop. The loop contains an unusual CG
base pair. As in the 14-nt RNA, all residues are rigid
at the measurement temperature (310K), except for residue A15 in the loop.
46
. _
.
. .
Chapter 2: F(HCi-Experiment
2.2 METHODS AND MATERIAL
2.2.1 Sample preparation
The uniformly 3 C, 5N labeled 14-nt RNA (5'-PO4-GGCACUUCGGUGCG3'; bold residues
constitute the loop) was purchased from Silantes GmbH (Miinchen, D). The concentration of the
NMR sample was 0.7mM in 20mM KH 2PO 4/K 2 HPO4 , pH6.4, 0.4mM EDTA and 10% v/v D 2 0.
The
30-nt
selectively
RNA
13 C,15 N
(5'- PO4 -GGCACUCUGGUAUCACGGUACCUUUGUGUG3'),
which
is
labeled in the G and C residues, was synthesized by Jens W6hnert by n /t
transcription with T7-RNA polymerase with a linearized plasmid DNA as template and purified as
described previously [12]. Unlabeled rNTPs were purchased from SIGMA (Taufkirchen, D); labeled
nucleotides were obtained from Silantes GmbH (MUinchen,D). The final concentration of the NMR
sample was 1.2mM in 10mM KH2 PO 4/K 2 HPO 4 , pH6.2 with 40mM KC, 0.2mM EDTA and
99.99%v/v D20.
2.2.2 NMR spectroscopy
2.2.2.1
F(HCN)-experiment
The NMR data were collected on a Bruker Avance 600 MHz spectrometer equipped with a 5mm
'H{i 3 G/l5 N} Z-Grad TXI CryoProbeT, an AV 700 MHz UtraShield
5mm 1H{ 3c/
15
T
instrument equipped with a
N} Z-Grad TXI probe, an AV 800MHz spectrometer with a H{' 3C/ 5 N} Z-Grad
TXI CryoProbeT
and an AV 900 MHz spectrometer with a 5mm TX[ H{ 3C/ 15N} XYZ-Grad
TX probe. The data were processed on a Silicon Graphics computer (Origin2000) using Bruker
NMRSuite
(XwinNMR
3.5 and
TopSpinl.1)
programs
and
analyzed in Felix2000
(msi).
Measurements were carried out at 298K for the 14-nt and 310K for the 30-nt RNA. The
experiments recorded for the different molecules are summarized in Table 1. The
sequences are given in the Appendix (Section A2.1).
47
(HCN) pulse
Chapter 2: F(HC
2nd
Molecule
Experiment
Field
Strngth
nucleu
Number of
M
SW
ppm]
s
Scans
complex
Plt
ref
cross
t1
t2
600MHz
15N
15ms
40
64
1024
128
1024
700MHz
15N
15ms
20ms
15ms
5ms
lOms
15ms
20mis
30ms
40ms
40
40
4
4
4
4
4
4
4
16
16
32
-
512
512
1024
512
512
512
512
256
256
100
100
42
30
30
30
30
30
30
1024
1024
1024
1024
1024
1024
1024
1024
1024
15N
13C
F(HGiICr)
14-nt
RNA
50ns
4
600MH-z
1
5N
15ms
40
64
1024
128
1024
700MHz
15N
15ms
40
16
512
100
1024
rF(H2' CQN)
600MHz
13C
lOmns
7
128
1024
50
1024
rFI'GCi'N)
700MHz
13C
5mis
lOms
15ms
6
6
6
6
-
1024
1024
1024
1024
32
32
32
32
1024
1024
1024
1024
6
6
6
16
1024
1024
1024
32
32
38
1024
1024
2048
5
5
32
32
1024
1024
32
32
2048
2048
F(H6/8C6/8N)
30-nt
~20ms
RNA
F(-6/8C6/8N)
900MHz
13C
30mns
40ms
5mns
800MHz
700MHz
13C
13C
5ms
5ms
Table 1: 7(HC)-expamc
2.2.2.2
-Experiment
arnia ot onide 14-r and de 30-ntRNA.
J(C,H) coupling constants
3J(C2, ,H,)
4
and
3 J(C6/,,H ,)
8 1
coupling constants have been determined using the pulse sequence
given in ref. [5]; experimental parameters were taken from the reference. The spectra were obtained
on an AV 700 MHz UltraShield T instrument equipped with a 5mm H{1 3C/1 5 N} Z-Grad TXI
probe with 16 (64) transients in both the reference and the cross experiment for the 14-nt RNA (30nt RNA). The evolution delay coupling constant was 47.6ms. The carbon carrier was set to 89ppm.L
The spectral width in the carbon dimension was 20ppm. 200 (128) increments were acquired in the
indirect dimension for the 14-nt (30-nt RNA).
2.2.2.3
Atocorrelated
relaxation rates
Autocorrelated relaxation parameters (R1 , R2 and the hetnOe) for C1 , and C 6/C
have been
determined as described in detail in Chapter 3. For the 14-nt RNA, rc has been extracted from a
48
Chapter2: F(HCN)-Experiment
detailed model-free analysis, while in the case of the 30-nt RNA, no extensive data analysis was
carried out at 310K and xc was obtained from the averaged R 2 /R 1 ratios as described in Chapter 3.
2.2.3 F-rate calculation
All theoretical cross-correlated relaxation rates have been calculated analogous to Eq. (1). For the 14nt and the 30-nt RNA rotational correlation times c of 1.9ns and 4.1ns have been applied, as
determined from the measurement of autocorrelated relaxation rates (see Chapter 3). The CS-tensor
values and orientations as well as the lengths of the respective dipole-dipole vectors used in the
different cases are summarized in the Appendix (Table A1).
All r(X) correlations involving the
1 5N
CS-tensor of N1 and N9 have been determined using C++
programming language. The coordinates of the
15N CS-tensor
components and the CH vectors were
obtained from a set of modified mononucleotides rotated around the angle
in the program
INSIGHT II. These coordinates were then put into a C++ program in order to calculate F. For
c-',
S' A
c
6D8,CSAN/9
and
rH6'8CSA9C618, coordinatefile independentcalculationswere carried
out using C++ programming language. A detailed description of the parameterization procedure is
given in the Appendix (Section A2.2).In the case of
rF'
iclsX
only the orientation of the tensor
components in respect to the C-H r . bond is known. The maximal possible value of
o
rH'DC
cl
has
been calculatedby rotating the tensor around the C.-H r bond using the program Mathematica.
2.2.4 RNA structure calculation
Structure calculations on the 30-nt RNA have been carried out by Oliver Ohlenschliger at the IMB
in Jena using the program CYANA [13]. The calculations comprised 937 upper distance restraints
obtained from nOe intensities, 735 of which were either intraresidual distances involving the I- 5 ,/H5 ,,
or interresidual restraints, the rest were restraints ensuring the circular ribose structure and hydrogen
bonds distances. In addition, 212 lower distance constraints were incorporated for ribose ring closure
and the hydrogen bonds for the base pairs within the stem-regions (1-13; 18-30). Furthermore, 118
angle constraints were incorporated: The backbone angles a and ( were constrained according to
49
Chapter 2: F(HC)-Experiment
phosphorous chemical shifts; 6, v1 and v2 were constrained to fulfill the ribose pucker requirements.
Xangle constraints
were directly incorporated as angles with a variance of + 10° . All up to 4 possible
X angles obtained from the degenerate F(X) parameterization were included resulting in a total of 38
X restraints for 10 residues. Out of the 200 structures calculated, the 40 best one (with a target
function lower than 0.47A) were selected.
50
Chapter 2: F(HCN)-Experiment
2.3 RESULTS
2.3.1 The quantitative F(HCN)-experiment
2.3.1.1 Pulse sequence
The pulse sequence and the experimental parameters of the quantitative F(HCN)-experiment are
shown in Figure 7. A quantitative approach has been selected, in which F'cDCSA is obtained from
the relative intensity of the same cross peak in two subspectra.
The experiment is a modified HCN-experiment
[14-19]. The magnetization transfer is given in
detail below the pulse sequence (Figure 7). CN-double- and zero-quantum coherence is excited at
time point a and allowed to evolve for a variable delay rM under the influence of r ooNcSAinto one
term, which is
b-modulated
h
by the F-rate (4HC/F,
NpsA
modulated term (2CNsin(HDcDcsA
*,)),
and into a second sin-
at tme point b [20]. Two experiments are recorded, the
*rM))
reference experiment, in which the tush-modulated operator is transferred back and the cross
experiment, in which the sinrmodulated
operator is achieved in an at-and-hik
operator is selected. While selection of the reference
manner, the specified phases (caption of Figure 7) have to be
shifted and an additional proton-carbon
recoupling period has to be introduced in the cross
experiment. Furthermore, a proton 90 ° pulse is applied after
remove any operators involving protons.
HDCSAN
TM
in the cross experiment in order to
can be determined from the peak intensities in
the two experiments according to Eq. (3).
(3)
r DDCSA= tanh -( nsf cro
HC'N~~~~~~
In this, nsd and sd
M
crossI ref
are the number of transients and
pr
and 1- are the peak intensities in the
reference and cross experiment, respectively.
In principle the F(HCN)-experiment
1
simultaneously, thus obtaining
can be applied to the base and the ribose moiety
CSA
.Hr CrN/9
],DS
and
HD6DI'CSAN1/9
at the same time. However,
sensitivity is improved by running two separate experiments, since the HG and CN-transfer delays
differ for the ribose and for the base moiety. In addition, the carbon carrier frequency can be set on
resonance, when running both experiments separately.
51
Chapter 2: F(HCN-Experiment
TROSY
~~~~~~~~~~~T
T.
t
T.
t
cross
~ ~~~ ....
~ _.~ ~' ~ ~ ~~=2T -A
rf+...................
I~~~~~
I * ,',-....~Y..................
cross~~~~~~~~~~~'"
2 ' = 2T . ...................
'
........
;, ' ,";
. ...
' ...';...
I
Y
.....
I
'H
I
WFII
15 N
G
1
+
rf cross
....
~
:
.~~
:
I
G2
.I
:
96
AIT-A
~
(P7
I 2CYH.S(F¶M)
I
~~~~~~~~~~~~~~~~~~~
C2'IC5
:
~~~~
PS ~~~~~~~~93
(Ps
a
&2
22
(N
(Ps
T-AATIAEAI
2C.N.S(Pr-m)
i~tiA
4
IC~
I4
G4 G 4
&G
I4
b
crs
,
"
Y
I
T2
cross~~
IC~srM
Hy
2
2-
I .I.
1
2C.N.S(FrM)
1221IiA *~~~~~~~~~~~~~~~~
II
:*
2
I ='.2Tii
13CTII
(Pi
cross
I
I
T
cross
2 2
1
I
HAsFTM)
GARPI
:~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
IefcrosszIyH
'
6
IC~SrM
MH
I I
I GARPI
G7
I I) lyll '-
Magnetization Transfer
ref
4CYNYHzC(F¶M)
H2CI~c
Figur
7' Qritatiw
-CNH
I(HCN)-expefrnen
4CYNzHzc(rTM)1JCH
2CYH.C(rirM)
HACFTM)
nNH
H,N
Narow and ide bars aconpr sto 90 and 180 degme pulses, respeatiy
Sdewpulses andgradi are iit
by semi-e.gses.Thedefat pulsephe is x. he ni e and e cras ex
t
aresnm zed in oe pulseseque7e sdm For nth, e rp nn phses 3, (p4am set to x,
is set to y and te
haed 180°0 pulselalxdd fis appliedonprm. In te oas ex nn ples
, epare set to:
(s is set tox andithe
hadsd 900° and 180° pulses lad cmss areappliedA sletie off-o t ptlseon C2 orCs drg cam am
otnt tine
enicalshf edaim is shom in gy A aq onSparanmet
for r o,cDC
(rH,C
"Ja
. A419/is e 1 tto 1/(2
9 t'A
6/
IC',NII1
H8C618,Nl/
12*1~
1
andsetto3.2ns (2.4n); T=1/(2*Jvc) is38ns (32r); r i the m
set to 89ppm(141pp) andthe rtgenet
Ci'/CC&:
1.77rs Q3 rasing
at
158ppm
At
, °DD
;CS
600MHz,
raxat
petc
Thecaa n iet
s
sd elaepulses mre
set asfdlos:
pulse [21]; Nl/N9.g: 1.7r IBURP2 imersionpulse [22]; C2 '/C 5. 512/1s Q3 pulse
Waterfliphbzk (WFB): ln
sr puse. Gradientas:
Gil=lrns, -20Gonl; G2 =1rns,20Gonl; G3 =1rs, -25Gonl;
1
G4=500ps, -2.5Gon; Gs=lrrs, 22.5Gon ; G6 =1ns, 2Gnm; G7=ns, 10.05Gan1 . Phase cydinr (0-=x,(-x);
(= 4y,4 (-y); q3=8x,8(-x)for he *e
exmr
; 8 y,8(-y)fir the ass exp
rmt (p4=16x,16(-x) in the e/m
expemrn, 16y,16(-y) in te crss expeit
=x,2(-x),x,(-x),2x,2(-x),2x,(-x),x,2(-x),x,(-x),2x,(-x),x,2(-x),2x,2(x),x,(-x),2x,(-x).Sates- TPPIphase disinition
[23] in oi
s tair by shiing phase =x,y Gradie ie
selcion in 2is adiezdbyshifgpases (6=2x,2(-x) and q7=2(-y),2ytiher uith dangigthesign fG 6. Asynom
GARP dling
Magtizati
[24] is sed to spss
tramferpadmay in the *men
TROSY.Eution
of the GH TROSY
her
de salarmplingsduig acqisitn Magnetization Tnsfe
('ref) and the crss expe
arnt
52
('crass). C0sh is abnuted
by 'c, sirnh
by '
Chapter2: F(HCN)-Experiment
Although the F(HCN)-experiment is shown as a three-dimensional sequence in Figure 7, it is not
meant to run as such due to the time requirements for the cross experiments, where at least 256
transients have to be acquired for each time increment in the indirect dimensions. Instead, depending
on the resonance dispersion, either the carbon or the nitrogen 2D plane or both have to be acquired.
It has to be noted that
3C
is preferable to '5 N chemical shift evolution, since the latter takes places in
a real time, while the former occurs in a constant time period. Real time chemical shift evolution in
general constitutes an additional time interval in the pulse sequence, during which in this specific
case the favorable TROSY-effect is averaged out by the 180° proton pulse.The total CN-transfer
delay (2T) constitute the longest period in the pulse sequence (62 or 76ms). In this time interval,
relaxation of the carbon transversal magnetization due to
CDCSA
cross-correlation with the
directly bound proton is significant[14, 19]. Depending on the spin-state of the proton, the dipolar
and the CSA component of the relaxation mechanism counteract (TROSY-effect;[25D)or add up
(anti-TROSY-effect). In order to minimize signal loss during 2T, the slowly relaxing CH TROSY
component has to be maintained. The evolution of the TROSY (+) and the anti-TROSYcomponent
(-) are shown in Figure 7.
While 180° pulses on carbon do not affect the TROSY evolution ('cDCSA ), since they switch
both carbon operators, the TROSY and the anti-TROSY component are interchanged by proton
180° pulses. The necessity to recouple protons in the cross experiment results in evolution of the
anti-TROSY component for a period A, while in the reference experiment the TROSY component
evolves for the whole delay 2T. The loss in signal intensity due to this additional relaxation effect in
the cross experiment as compared to the reference experiment has been estimated from the
of the
calculation
s6/8,N1/9 to be less than 1%. In this calculation, carbon CS-tensor values
determined by Stueber et al. [8], a field strength of 600MH-zand a ; of 1.9ns and 4.1ns for the 14-nt
and the 30-nt RNA, respectively, have been used. For C1 ,, this contribution is even smaller than for
the base moiety, since the CSA of aliphatic carbons is significantly smaller than the CSA of aromatic
ones. Therefore, this differential relaxation effect in the reference and the cross experiment has been
neglected altogether.
The reference and the cross spectra of a
13C
2D-F(HCN) applied to the ribose moiety of the 14-nt
RNA are shown in Figure 8. The experiment has been run at a 700MHz spectrometer within about
15h. The experimental sensitivity is sufficient to yield an error in the resulting F-rates of 0.1Hz on
average.
53
Chapter 2: F(HCN)-Experiment
The relaxation delay rMhas been optimized for maximum signal intensity in the less sensitive cross
experiment, which picks up the srimodulated
proportional to sirf£*ru)
cross operator. During
TM,
signal build-up
is counteracted by signal loss due to autocorrelated relaxation of the CN-
double- and zero-quantum coherence.
A
B
[ppm]
U7.
-
c8
13
[ppm]
-
c
-87
13
c
-87
-88
-88
0
a
-89
-89
-90
-90
L.
G12
A4."eC14
G2
S
G9
6.0
U11
·
eC5
C3 C13
OS
*
I
5.0
4.5
4.0
'H
-91
*
-92
U6
5.5
GlO
-91
G10
6.0
[ppm]
-92
5.5
5.0
4.5
4.0
'H
[ppm]
Figure 8: -C 2D-(HCN)-spectra ofthe ribosenietz
f the 14-nt RNA. (A) ROfem
exennmng(B) aurs expem-m
the semiisdty in the tzeo expernn.
he
spectrahae been rmmd on a 700MHz speamvnrter,rM wtsset to 15nr;the nmm epXennr 'tws ! *aiwth 32,the
Resonaxr assignrn
rsseemnv
aregin
D-slices drmgb the reswme ofGl O denmtrate
wth 1024 tramiens.
For the ribose moiety, cross peak intensities of representative residues (Figure 9) show that a rMof
20 to 30ms is optimal for both the 14-nt and the 30-nt RNA.
Due to the larger chemical shift anisotropy of the aromatic compared to the aliphatic carbons,
which constitutes the main difference between the two moieties, optimal evolution delays for the
1F(HCN-experiment applied to the base are expected to be of the same order or slightly shorter.
54
Chapter 2: F(HCN)-Experinment
14-nt RNA
30-nt RNA
"7
h(d
-
.Fa
0
, G1
0
041
043
-0
§
0.03
044
.T
"~ , ~
..
t
-
r
0.05
i
0
--t~~~~~~~~
C14 0.ILCD
75G.....
C,4
4tM [S]
TM[S]
inte 1(HCN)cross
ex mnta o e
Figure 9:Peakieoiti i d dn
ffe edu im delayrMf seareortos
arnd tpek ierites arisefmpncsze or W e
ri0e nrietyfor the 14-nt(left)arndthe30-ntRNA (ri. Pacsitizw
sig orthef -rates.Data hasben arat
700MHz ihzb
512 (256) traraie foreab tr imrntfora rm fC5 to 30n
(40 to 50rrOns)
for tdxe14-ntand 1024 trarmients
fr all zm ddasfrte
azewraged
noiseleze
30-ntRNA.
The er
rs hae ken takenfimt
2.3.1.2 Derivation of F(X) parameterizations
For all F-rates, F(X) parameterization curves had to be derived. These
(X) parameterizations
follow Eq. (1). Hence, isotropic diffusion as well as the absence of internal motion (S2=1) is
assumed. CS-tensor values and orientations reported for adenosine, cytosine, 2'-deoxythymine and
dihydroguanosine in the solid state [8] are used. The N1 CS-tensor from 2'-deoxythymnine has been
used for uridine, because no tensor for this nucleoside is available to date. The global rotational
correlation time tc has been obtained from autocorrelated relaxation rates (R1, R2, hetnOe; see
Section 2.2.2.3 and Chapter 3).
TcDDCSA
,H,.N
(*)
__
_A__
Adenine
Cytosine
Guanine
Uddine
=
=
10_
o [Alcos(X)+ A2 cos(2*) + A3]
A
A2
-5.933
-7.137
-6.828
-6.860
0.332
-2.123
1.265
-2.908
Table 2: Famerexparon of de depo ef
tn' Bo= rmgcfiddsmt
in Tesk
r Hicl,
9
nZfor
55
A3
3.338
4.059
3.271
2.917
d7e
mdeaides. rc=ozeralrtatnl
wndation
Chapter2: F(HCN)-Experiment
In the case of r
, the parameterization is a straightforward application of Eq. (1), which
IooC1
has been expanded into the nucleotide specific Fourier series shown in
Table 2 in order to facilitate the angular interpretation of the F-rate. These parameterizations
require only to incorporate the rotational correlation time of the investigated molecule and the
magnetic field strength of the spectrometer.
Nucleotide specific F(X) correlations for the 14-nt RNA with a correlation time of 1.9ns, are
shown in Figure 10. For all nucleotides, the slope of the F(X) correlations is steep in the most
Figure 2) combined with a
frequently adopted range of the most populated anti region (180-210°;
sign change, offering a sensitive parameter for X. In the most populated
swnregion, however, the
parameterization curves are flat, indicating that angle determination will be less precise in this region.
At closer inspection, it becomes apparent that the results of the F(H-CN)-experiment are not as
easy to interpret as expected. A more thorough analysis reveals that in addition to F ,cSA
dipole-dipole, CSA contribution has to be taken into account, namely Fr
Adenosine
N
6
I 1.5
I
5
4-R
0.5
7
2.5
6
1.5
5
0.5
4om
3
-0.5
-1.5
1
-1.5
-2.5
0
3610
-2.5
90
180
270
3
Q_ -0.5
2
0
2
LL:
0
90
Cytosine
2.5
6
Ir 1.5
5
? I
A
-0.5
I
4om
2.5
6
1.5
5
-1.5
1
-1.5
-2.5
0
-2.5
180
x, II]
Figur 10:Nudect spea r
idis
270
7
4.m
o~ .. 0.5
Qi. -0.5
QO~ .0.5
3
2
90
360
3
2
1
0
90
swsryhon
180
270
0
360
Xo1
CsA (,)
parane
ionaes fr the 14-nt RNA at 600MHz. The ane
in the ag riboas subuit [2] are shen in gny ThenMst
also in
0
36 0
8
3.5
7
0
270
8
3.5
0.5
0;2
180
1
[]
X [0]
pims
8
3.5
7
2.5
'CSA
Guanosine
8
3.5
asecond
zamhizgbibW
56
.. .... -
.
a anle gom ite
an andfordx
Chapter2: F(HCN)-Experiment
Since rFoo
cSA and
"HCN
rF'HN ,CSA
,C
arise from interactions involving the same operators, q, Hz and N,
(for the former [[2qHz,[Nz,p]]; for the latter. [[2NzHz,[q,p],
they cannot be separated
experimentally. In addition, the evolution of both cross-correlation rates results in the same cross
operator. For the ribose moiety (Figure 11A),
rFDD,CSA
is small due to the small anisotropy of the C,
CS-tensor, which is around 30ppm in the C3-zo and about 60ppm in the C2-endoconformation
[26]. Although no complete orientational information can be obtained for this tensor to date, the
approximate contribution of
to the overall rate has been calculated from the C1, CS-
rNJ,9,cl
tensor values and their projection angles on the C1-H1 bond provided in ref. [14]. Since this tensor
displays an anisotropy of around 60ppm it can be used to judge the maximal possible contribution of
FD,IcSA'
For the 14-nt and the 30-nt RNA with a rotational correlation, time of 1.9ns and 4.1ns
the maximal contribution of
rFJ'DCIsA,
amounts to only 0.06 and 0.12Hz, respectively.This small
contribution has been neglected in the following analysis.
OH
.v-vv.r
OH
A
l-~
~~DD
~
.CS
,c (4 aMTH
alr~~~~~'
Figur 11:
(iHt/N1/9c/8(B
9C618
B
.CSsctrAw f"the
(B).The '3C
3 SA and the leftdN-
For the base moiety (Figure 1lB), nucleotide specific rD'
based on
13 C
H oraeg
/ 9 C618 values have been calculated
CS-tensor values taken from Stueber et al. [8]. The results are shown in Table 3 in
comparison to
fD,CSA
I6/sCf6/8N1/9
-rts r'o
"'i~rsieDD.A
Since the two F-rates are of similar size,
neglected.
57
H6I8NI
9
C6/8
cannot be
Chapter 2: r(HCG-Experiment
DD,CSA
r
H6/8N1/9,C6/8
[Hz]
-0.60
-0.76
-0.59
-0.78
Adenine
Cyt:idine
Guanine
Udine
Table3:
3: F,
r'DD, CSA 6 I 8
Table
n68Nl19,C618
6
a
DD,CSA
H6/8C6/8,N11/9
[Hz]
-0.91
-0.45
-1.46
-0.17
fr
for d''e dA
10E~
dr D,CS,4
FI6/8N1/9
"n618C618,Nl<9
fian
a "N CS-15NaMi
badOni13C
3CagsNC-edTo$
~
n~""
Studeret aL [8]for the 14-ntRNA at 600MHz.
If both r'HpDcSAand rFHDNDcsA
have to be taken into account, the evolution of the operators during
TM differs
from the one discussed in Figure 7 in the presence of just one cross-correlation. In this
case, the reference (Eq. (4)) as well as the cross operator (Eq.(5)) evolve with the product of the two
F-rates.
4H AI h(
4HzCyNpoh(
(4)
rDD
(5)
c
DD ,CSA
,N,CSA r)) csh(f
HN,C
)
M)
sA *rm)
sinh(DD'cSA*)
~4CNsinhb(F
The ratio of the cross peak intensities in the reference and the cross experiment can be simplified
to Eq. (6):
I cross_sin[(FHCN +FHN,C
)*T ]
(0/
Irf
For small values of
lim(cosh(x))=l
TM and
cosh[(FHCN rHNC )*T]
FHCJ
- and
FHDS
i-rates
of similar size, lim(sinh(x))=x and
apply. In this case, Eq. (6) can be rearranged to Eq. (7):
X-0O
Icross
(7)
Iref = (FHC,N + HN,C) * TM
Using a typical TM value of 0.02s, this approximation is accurate within a 1% error margin for
(-S
HCN
"NDcS ) values of up to 7, which correspond to a correlation time of about 8ns for
guanosine, 1ins for uridine, and 22ns for both adenosine and cytosine. Hence, for RNA-molecules
which is the
of up to about double the size of the 30-nt RNA, Eq. (7) can be used to obtain F-hbase,
58
Chapter2: F(HCN)-Experiment
sum of the cross-correlated relaxation rates,
FJCDNSA +-vl
s
.NC
-hse parameterizations for the
different nucleotides are summarized in Table 4.
7
Ftse=10
BorCA4
A4
Adenine
-5.105
Cytid&ne
-4.057
Guanine
Udine
-6.922
-3.206
Table 4:Paramwnzation
r F base
Consequently, the ratio of r
cs
and F-ase yields the
c and B independent F-ratio()
relations given in Table 5.
T - ratio () = 107 [Alcos(*) + A2 cos( 2Z) + A3 ]
Adenine
Al
-0.065
A2
1.162
A3
-0.654
Cytidine
Guanine
0.523
-0.183
1.759
0.986
1.000
-0.473
Uddine
0.907
2.140
-0.910
Table5: Fner epamnin fhdepdgf he F-ra onfor th difr e m
2.3.1.3 Determination of X from rH ,csA1
-D,CSA
FHDDcSA
/9-rates
have been extracted from the peak intensities in the cross and the reference
spectra of the F(HCN-experiment
as described in Eq. (7). The measured F4D"cIslc
-rates and
resulting X angles for the 14-nt RNA are listed in Table 6 together with the reference angles from the
crystal structure. Also given are
angles extracted from the measurement of 3J(C,)
coupling
constants.
The precision of F-rate determination is 0.1Hz as obtained from the mean deviation of the F-rates
for all residues from multiple measurements at different field strengths and different relaxation
delays rM(see Materials and Methods).
59
Chapter 2: F(HCN)-Experiment
r DD,CSA
1Hl'CI',NI/
Residu
,
9
[H][Hz]
Residue
F-base
"
Hcr
DSAN9
F-tio
X[ I
3 J(C,H)
Refemrence
G1
G2
C3
0.67 + 0.04
0.14 _+0.01
-1.16 + 0.18
-0.99 + 0.16
-0.66 + 0.00
203 + 1
203 + 1
192 + 4
201 + 1
192 + 2
-
194.8 + 4.4
194.8 + 4.4
198.4 + 3.1
A4
0.44 + 0.01
-1.30 + 0.11
205 + 1
204 + 1
198.5 + 1.5
198.2 + 3.5
C5
U6
U7
C8
G9
0.32 + 0.07
-0.29*
-0.99 + 0.11
-1.03 +0.08
-1.64 +0.35
-0.68
-0.56
-0.35
-1.00
-1.92
+ 0.05
+ 0.04
+ 0.10
+ 0.00
+ 0.20
200 + 1
202
212 + 2
221 + 2
60 + 8
197 + 2
205 + 1
240 + 9
226 + 3
60 + 7
191 + 8
196.5 + 4.5
217.5 + 9.5
210.5 + 6.5
93.5 + 4.5
197.9
211
223
202.6
58.7
G10
1.00 +0.11
-1.09 + 0.08
198 + 2
184 + 4
215.5 + 13.5
198.3
Ull
0.29
-0.30 + 0.04
194
187 + 1
-
198.2 + 3.0
G12
C13
0.53 +0.04
0.21 +0.04
-1.10 + 0.04
-0.80 + 0.15
206 + 1
202 + 1
198 + 2
200 + 1
192 + 4
189.5 + 3.5
194.8 + 4.4
198.4 + 3.1
C14
-0.55 +0.08
-0.85+ 0.01
212 _+2
216 _+2
198 + 5
198.4 + 4.4
6.8
13.1
7.2
14.7
8.3
9.7
*
p DDCSA
RMSD
DIN/,r
_,_csA
6.8
13.1
F-Mtio
3J(GH)
[0]
' ° c
Table 6: rH~I'CI',N
s
19
xang
and F-baseforthe 14-nt RNA.
HI1',N,/
14.7
rsultingfromtheaml)sis ofalone
DrFo,1/9 andfirm the
ratio of the to Frates are cpared to the arns fiom the cstal striazure(oldm deatd Referx) andfrom the
at 600 andat
Fratesfirn nasens
interpmtation
f3J(CH) ortplingcwtants (almn denoed'3J(QH)). Theazeraged
ty pecspcKarplus700MHz aregiwn 3J(C,H)axling constants
haze n demein and analyzedby a refir mdeide.
relationas
describedpziosly[4-6].Aeragedanglesfimtheanalsis of3J(C2/4,Hl)and 3(C6/8,H1) aregi E rro for
TM edution pmios and dffrent rmg
fields(see
the F-rateshae bn caladated
firn mltiple nraswrnts at diffemvnt
differns heeen angle
Materialsand Metdls sectio*; ers for coling constantdenizedX anles hawebeentaken fim thedx
detminationfltyn3J(C2/4,Hl)andfom 3J(C6/8,Hl).
For both F-rates and J-couplings, only the X angles closest to the reference values have been
extracted from the degenerate parameterization curves. The agreement between the X angles derived
N'IN/9and the reference angles from the X-ray structure, RMSDrF(CHN)/x-ray, is 7.2 °.
from 'r' D-Do,CSA
This agreement compares to the one between the reference angles and X angles obtained from the
analysis of
(RMSD,
3 J(C2q/,H r)
4 1
J(C ,H) IX-ray
=
and
9.70).
3J(C6/ ,H r)
8 1
coupling constants, which are also given in Table 6
The consent between the J-coupling and the cross-correlated relaxation
derived X anglesis slightlyworse (RMSD,
J(c H)/r(HCN)
=
13.1°).
For cross-correlated relaxation rates as well as coupling constants, the deviations from the
reference angles are significantly smaller in the stem-regions
60
(RMSDr(CHN)xray
(stem)=6.0°;
Chapter2: r(HCN)-Experiment
RMSD3 J(C
H)/X-ray
(RMSDr(CHN)/X-ray
(stem)=4.4°)
than
in
the
non-canonical
loop
region
(loop)=9.9°; RMSDJ(c, H) / X-ray (loop) =17.1°). A major contributor to the high
RMSD of the coupling constants as compared to the F-rates in the loop region is residue G9, which
is in the sn conformation. Whereas X is 58.7 ° in the crystal structure and 60 ° from the analysis of
cross-correlated relaxation rates, the coupling constants result in 93.5 °. In contrast to differences in
precision for different secondary structure elements, no base type specific differences can be
determined within the limited number of investigated sites.
2.3.1.4 Determination of
from F-matio
F-ase values of the 14-nt RNA are listed in Table 6. In Figure 12, experimental F-ratio values are
plotted against the reference angles together with the theoretical parameterizations of the individual
nucleotides. The X angles resulting from F-ratio are also given in Table 6. The overall agreement
between these and reference angles is RMSDr,,
x.ay=
determination is the same as from the analysis of rHD'c,cl
determination methods proposed here is RMSDra./x,,
8.3 ° . Hence, the precision in angle
alone. The agreement of the two angle
= 6.3 ° . As for rDDCSA
the precision is
higher for the stem residues (RMSDrraio/ xray(stem) =6.3° ) as for the ones in the loop
(RMSDFratio/X-ray(loop)=11.9°). For both methods,
Xof G9'Ynis 60°, since the measured value
exceed the values in the parameterization curve and 60 ° is the angle in the syn region with the
maximal rate. However, subtraction of the error of the measurement yields values within the
parameterization. Figure 13 shows correlations of the
X angles derived
from r'oocsA
alone and
from F-ratio compared to the reference angles. Although there are differences in the deviations
from the reference angles, no systematic differences can be detected between the two techniques.
61
Chapter 2: F(HC)-Experiment
3.5
2.5
1.5
2
.- ' 0.5
-0.5
-1.5
0
180
90
270
360
Xl
Figure 12:Exprna
paraeatm.
F-ratio
pkateagist themfwe al andthoica Fratid,)
i fr the 14-ntRNA
F-ratio
"
250
aa
ADU
230
230
b
o
0
_ 210
f 210
190
190
190
170
170
170
190
210
230
250
170
190
Xref[]
Figume13: Xargz dtdfiTn
Lop residuesare lll
by bces. X e
210
250
230
Xref ]
I
9,cl, (lft) andF-ratio
(riit) twpandto ew an
for G1 arenimsis sime rHF
62
9,C,
=
not be
for th 14nt RNA.
'vfr
esid
Chapter2: F(HCN)-Experiment
2.3.1.5 The N CS-tensor in solution from F-base
Figure 14 shows F-base obtained for the 14-nt as well as the 30-nt RNA sorted by base type
compared to the theoretical F-basevalues.
-0.5 -1.0 ±
-1.5 -2.0
8
.
(cI
c ct
*+ +
14-nt RNA
0.0
]
.
+
½
+
-
.
---
4 3 5
-2.5
A
Q 1
W I'
4A
I39;i 1
-
2 9 1012
C
G
6 7 11
U
30-nt RNA
0.0
-1.0
N
-2.0
4~~
'T'
I:
-3.0
co,
Q
-4.0
-5.0
-6.0
-7.0
374162122301
5
-3
2 9 10182
5 7 1416212230.1
2 9 10182628
.
G
C
Figue 14: Expenntal and theoreia ults the (HCN)-exnt
appliedto the base nrety ofthe 14-nt (to) and
the 30-nt RNA (botorr sortedby base type Gddated F-base luesare indicatl by dashedhorizonta1lires. Lop residuaes
are laid
zdthbocesand hghlightedby greyshading For the sdectidy laded 30-nt RNA, F-basezaluesare gizn onlyfor
the 13C1SN lalde guansi ad cytosimresids. The datafor the 14-nt RNA has beenobtairndat 600MHz, the onefor
the 30-ntRNA at 800MHz.
63
Chapter 2: F(HCN)-Experiment
Since -baseis not dependent on the conformation around X,comparison to the theoreticalvalues
allows to obtain information about the characteristics of the N 11 9 and the C6/8 CS-tensor in
oligonucleotides in solution. Unfortunately, no individual information on either tensor can be
obtained, but only their combined effect on F-base can be interpreted. From this data, the following
conclusions can be drawn about the CS-tensors in the bases of RNA:
(i)
They are base type specific. In the stem regions, all the adenosine, cytosine, guanosine
and uridine residues of the 14-nt RNA and the cytosine residues of the 30-nt RNA
exhibit similar values for each base type. These canonical F-base values are, however, in
general not in agreement with the theoretical F-basevalues. Apart from the cytosines in
the 30-nt RNA and U7 of the 14-nt RNA, the theoretical values are in better agreement
with the loop-residues than the canonical stem residues.
(i)
They are correlated to the theoretical values. The relative size of the stem adenosine,
cytosine and uridine residues in both molecules matches the relative size of the
theoretical values. For guanosine, the theoretical value matches the value for G9 in the
syn conformation.
(iii)
They are conformation dependent. All the loop residues (except for U17of the 14-nt
RNA and C16 of the 30-nt RNA) exhibit cross-correlation rates, which are significantly
different from the ones in the stem-regions.
The first observation is intuitive, since the nuclei are surrounded by a different chemical
environment in the different nucleobases, and in agreement with earlier observations [8]. The failure
of the theoretical F-basevalues to reproduce the experimental values of canonical residues is contrary
to the better fit of the experimental X angles in the canonical as compared to the loop regions.
The second observation is expected, since CS-tensors of the same compound
environments
in different
should exhibit at least similar characteristics. The last result concerning the
conformational dependence of CS-tensors is still subject to debate in the scientific community.
64
Chapter 2: F(HCN-Experiment
2.3.1.6 Transfer to larger systems
The cross and the reference spectrum of the F(HCN)-experiment
applied to the 30-nt RNA
(Figure 6B) are shown in Figure 15. The spectra were obtained at a 900MHz spectrometer using
1024 transients per t 1-increment.
A
B
[ppm]
[ppnm]
13c
13
-nC
87
-87
88
-88
89
7 VW
G2 G28 C721
0o0
-:C21
G1
0
. . ..I
-91
C30 *C5
.......
6.0
C
A
*
C14
7- u
V
0 C22
G17
.
-89
I
91
2,
C22
-92
-92
I
I
5.5
5.0
I
I
.
'
4.5
4.0
1H [ppm]
I
.
I
I.... I
6.0
.
I
.....
' I
5.5
.
.
.
.
.
I
.
5.0
.
.
.
' I....
.
4.5
'H
Figure 15: (HCN)-spearaf the30-nt RNA. (A) RqOfr exp'n
.
.
I
.
.
4.0
[ppm]
(B) arss cpeinm Resonanxassigma are
giwn The spectrahawebeen aEd on a 900MHz spea xte rM ms set to 5ns; the ,b
expe
pme
Usdai
m
32, the crss expenn teih 1024 trarsient; The oerall duration the ttm expent r
s abwt 15 hotins.
FJif4CSA
th
F-base and the extracted X angles are given in Table 7 together with X values derived
from the analysis of 3J(C,H) coupling constants and the reference angles from the NMR-structure
[11]. The accuracy of X angle determination is 11 when derived from rHD4
oc CWs
alone and slightly
better (RMSDF ratio/NMR= 8.6) when the F-ratio is used. Angle values obtained from 3J(C,H) coupling
constants are in good agreement with both the reference as well as the cross-correlated relaxation
derived X angles.
65
Chapter2: F(HCN)-Experiment
Residue
-
Fbs
]',DD CSA
'~H1/9,F
[Hz]
[N1/9
Hz]
[Hz] C
-3.95
-3.06
-2.59
-2.56
-2.74
+
+
+
+
+
-4.29 +
0.28
0.03
0.22
0.04
0.34
0.33
FH
DD,CSA
208 +
190 +
189 +
212 +
FJ(CH)
I'-d0
205 +
191 +
189 +
212 +
-
G1
G2
C3
C5
C7
G9
1.17
3.21
3.57
-1.59
-
G10
-
-5.17 + 0.25
C14
C16
-2.05
-
-3.99 + 0.14
-3.07 + 0.16
G17
-1.91
-
G18
-
-2.27 +0.12
-
-
C21
2.43
-2.80 + 0.20
194 + 1
C22
3.38
-3.24 + 0.17
190 + 1
G26
G28
C30
0.75
-1.49
-5.98+ 0.17
-
-
-3.12 + 0.26
-2.93 + 0.80
210 + 1
211 + 1
rDH
'N1/9
,CSA
J(C,H)
C/SA
215 + 1
211 + 1
-
TZ) dest
x anglesfor 13Q 15N lied
F-baseand resdulting
to the refer
194 + 4
194.5 + 4.5
191.5 + 4.5
201 + 9
209.5 + 2.5
193.6 +
204.3 +
201.1 +
206 +
198.7 +
195 + 0
196.7 + 1.5
197 + 4
202.5 + 10.5
201.6 + 2.2
197.8 + 4.9
angle is shozen Angle defatic
0.5
0.9
1.3
3
1.6
195 + 1
200 + 8
199.8 + 1.6
193 + 1
194 + 4
197.5 + 1.6
209 + 1
211 + 2
222.5 + 14.5
200 + 3
208 +4
196.8 + 0.9
193.2 + 0.7
211.1 + 1.5
1.4
8.7
11
7.6
8.6
1.4
8.7
MR-
structure
65.2 + 1.1
7.6
7.5
ancire an cytcineresidues f the 30-ntRNA
wnTamred
to the X anglesfiman the NMR-struawre [11] andfroirm the intpretation
sdution for
0
97 + 5
85 +
F-rtio
[0]
1
1
1
1
-
p1DD CSA
R SD
Table 7. F
1
1
1
1
[
f 3J(QH)-capling constants.Ornly thed
for the Frates zeerecaladated using thedx
aweraged
RMSD f O.1Hz as determirdfor thedx
14-nt RNA; deziationsfor 3J(QH)-.derizeda angle
recalated from thedeziations
fangl resultingfm dxthe
analyisqfJ(C2/4,H1) and3J(C/8,Hl).
2.3.1.7 Incorporationof
rD'cs'
,c, into RNA structure calculation
In order to evaluate the structural relevance of the X angle restraints derived from r
Dc Ac1
they
were incorporated into a Distance Geometry/Simulated Annealing structure calculation in torsion
angle space of the 30-nt RNA. The calculations were carried out by Oliver Ohlenschliger at the IMB
in Jena, who had previously determined the structure of the 30-nt RNA by NMR [11]. For this
structure calculation, an exceptionally large set of nOes has been used (- 27 nOes per residue),
resulting in a very well defined structure (RMSD 0.67 A for all heavy atoms).
For comparison, two calculations have been performed, one comprising only a minimal set of
easily accessible nOe restraints (with the exception of nOes involving the H5 /H5,, protons, all
intraresidual sugar-to-base and sugar-to-sugar nOes (225 in total) were removed) and a second one,
in which the F-rate derived restraints were added. In this case, X angle restraints were directly
66
Chapter2: F(HCN-Experiment
incorporated as angle values with a variation of 10 degrees. All possible angles from the F(X)
parameterizations were considered.
_FDD,CSA
Cl 'H1'N1/9
+IFDDCS4
CVHI '11N119
Figure 16:Resultsf the CYANA sctme cadati on the30-ntRNA. A bxze Z ane di
guxne midua,for uhicha r HICI',NI/9
DDclCsA
cd be abta4 esdidtigfia*n
dx stmcue aklation
na whes(se tct). BdoezX ax c&isttims muigfine
and Z ange retrnts fm
fon
a
?igS
j
9 6tie
and
a ninmil set +
stmut aldati ii
the an3sis ef rFgD9csAI
9 . In boh caes, Z arl frn
1IC',NI/
a mil set nCe vudp
te 40 stmirms thd louest taet
Ysfirtan dx aalaoemaie of200stres areshon
Inclusion of the
r'
oCsA1 9 derived X angle restraints results in a decrease of the mean RMSD
from 6.9 ° to 2.6 ° for residues G2, C3, G5, G7, C14, C21, G28 and G30 (Figure 16). For G17, which
is in the syn conformation, the nOe based structure calculation results in a X angle of 67.5°+ 3.8° .
Upon incorporation of the F-rate derived X angle restraints, two X angles become possible, 42.8 ° and
79.6 ° , signifying that in this angle region, nOe and F-rate data are not sufficient to discriminate
between the two possible angles derived from the F-rates.
67
Chapter2: F(HCN-Experiment
2.3.2 The quantitative F(H2'C2'N)-experiment
2.3.2.1 Pulse sequence
(Figure 17), the first INEPT module is used to create
In the quantitative F(H2'C2'N)-experiment
C1
H 1,C.-antiphase magnetization. During the following delay T, magnetization is transferred from Q,
simultaneously to both C2 , and N1 (pyrimidines) or N9 (purines). In addition, the H1 , operator is
refocused during a delay A. At time point a, the double- and zero-quantum operator 4C,,,C 2,yNy has
been
During
created.
rH2%C25,sAN9
TM,
is
and
active
operator
this
evolves
2',.snh-HfC2',N1at time point
,C2,Nxsinh(
*t, and 8H2,,zC
4C,,zC2,yNycosh
( r Doc25a
22'NII9
'
into
b. In order
to transfer the latter operator back in the cross experiment, H 2 , proton magnetization is refocused
during an additional delay A subsequent to b, while the reference operator is transferred aot-and-li.
cross
ref
Y
1H
2
!
2
*
qX,
I|
3
I
DIPSI-2
DIPSI-2
*'P.P2
22
(Ps
2LY'
a
A
A
b'
l TI#tIA
~15N ''4`1~
O
2
9)6
I GARP
A2I2
I?
(p4
(P3
GG
T|
2
IGARPI
'
*
:
:G
3
A:
Gz
Magnetization Transfer
:
CH
ref
HYCH6-2CH
H;94
I
2
C JCNH
JCH 2.YH,.Zc(rTM)
4C..C 2.,yNyC(FxM) 4ClC.'N,c(FrTm)
2
s4
:fD4CH
C1C2YNY
Jcc
lj___)'
N
8C1 .C2 ..N.H2 .s(FrTM) 4C1 c2,N.s(F'TM)
cross
Hlc(rM)
2C1 .YH,,s(FrM)
Figur 1: Qnitaize 1(H2'C2'N)-exriPYn Tuoexpmnts, dx ire arl cossocpenrtmam
Hlys(Frm)
ndaeIn the
[f In
ee expennPmphae p4 is set to x andpon derqing dui te b& transfiris appidfor heperiodlabie
eiodlad cmross.A wterflohx pulse is appliedaftr
thecrossexcperin phase (p4is y and prm are detledafor the
eefzrstINEPT-step.Experntalparamten: A is "u to 1/(2*'Jca) andsetto3.2r; T=1/(2*1Jo) is38m; zm is the
2.5Gonl; G3=lrn, 40Gon'.
c=l/'Jcc-M Graes: Gl=lns, -20Gonl; G2=5001s,
ffcsNl/ 9 datwpel
2'HfC2',N119
aa pa
r=1
-mGad
.
ie epeni
(p4 =4 y,4 (-y) in & cross
Phse cding
V=x,2(-x),x,(-x),2x,2(-x),2x,(-x),x,2(-x),x.In adktion 6 is zsn t in
expennrt ps=32x,32(-x);p6=8x,8(-x);
in de co dinrmionMagnetizadtion Tmnsfer. Magrizatin
to aie quadraue detection
a State- TPPI[23]rrnurmr
ihin diepulse
('rf) and crss ('mrs) expewrnt at de iicated tim pos
and operatonsin the mfe
traferthmy
(
=x,(-x);
(02=16x,16(-x);
3=2x,2(-x); -4=4x,4(-x) in the
seqncz
68
Chapter2: F(HCN)-Experiment
Scalar interactions between C2 , and both C1. and C3 , are suppressed by setting the total period in
Extraction of
which C2, is transversal to 25ms (+c=1/'Jc).
\MCJLLJ~~~~~~~a
quantitative F(HCN-experiment
is similar to the
r-,FDC2SA
H2C2,NI9
(Eq. (7)). In case of the quantitative F(H2'C2',N)-experiment
no
optimization regarding TROSY enhancement or MQ was carried out. Since in agreement with the
the observed cross-correlation rates were higher than for the
parameterization for r DDCSA
H2C2',Nl/9
parame~~~~~a
F(HC)-experiment, no further optimization seemed necessary.
2.3.2.2 Resolving Xangle ambiguities with
H2C2N19
DDCA
F~~~~~~~~1"D
Figure 18 shows that in the case of C,2-edo ribose puckering
independent
offers a second,
H2'C2',NI 9
parameter, which potentially allows the removal of the ambiguity in X angle
determination resulting from the interpretation of
rFD',csII
9
alone. In this case, the actualvalue for
X can be unambiguously determined by virtue of the best angular agreement between the two
9 , and rF'C s1A1
parameters. In contrast, for Cy3-edosugar puckering, rFlc`s
but s"H2dC2',NI/9
sign
are of opposite
9
sign but show a similar F(X) dependence.
t3-enao
4-
4
'-pDDCSA
C1'H1',N9
3
I
2
7
.72
MIq
o-2
I
-,- .... ~~~~~~~~C2H2N
.'-''
''-"
-2
-2
r
- D D' C S A
'~C2'H2'N9
-3
0
90
180
270
0
360
180
x
x[Ol
Figur 18: angledepXnia
in a
(lid) rilse ecfmti
90
270
[0
I/ 9 (ds hlD)t for C2'eN (eif)and Cen9
rT
nded aresiiLr
Theparamnzationcuwfr theceher
NllCS
9 (s i
s) ad
69
360
Chapter 2: rF(HC-Experiment
r
DD,CSA
Residue
19
Residue F,~~
H2'.m/9
[Hz]
-2.3
-1.64
____7_
Cs
C2:
212 +2
268 +2
Z7
[0
]
223
237 + 1
221 +2
259 2
202.6
ZC'HI 'N
302+1
e71C
te (H2'C2N)-expn
fre
sbmisU7andC8 he 14-ntRNA.%cvnHi:T. uJCI 9 d,
emrshae
w n cxnrfimne aeraw derr
CCsAI9 -deiudX at*&;Zx, nivem , a*. 7The
Table 8:Resdts
Xanl;
%JW
ZC2H2'N
[0]
246 + 1
290 + 1
r
in Ff 0.1Hz asd
mirdfitnF
andrC6/8H6/8
'N9
1,
,Nl/
Table 8 shows the results of the F(H2'C2',N)-experiment
for residues U7 and C8 of the 14-nt
RNA, which are in C2-endo conformation, together with the resulting X angles and the reference
angles. Both F-rates alone result in two possibilities for
. For these two residues, the
parameterization and the experimental values for both
cNI N1I/9
rDI'
9
FH2'C2,
and rD,'%'
IC',N19aesoni
1 1 9 are shown in
Figure 19. The best agreement between r' ooAcISA - and
rF'JD
AsI 9
H2'C2',NI/9
-derived angles for U7 of 22 is
Figure19.
'e
bes agrement
etwee
CI,NI/9
THI
"
not in the region, where the reference angle is found. In this region, the agreement of the two
experiments is 'only' 34° . Whereas rFHDo,fcs
"H'C',NII9 results in a X value lower than the reference angle,
is larger for
rHD2DC',N9sA .
X
In contrast, for residue C8, the best accord of the two experiments (16 ° ) is
found closest to the reference angle. Judging from the error of the experiment of about 2° , this
difference is significantly smaller than the second best agreement (22°). It has to be noted that for
this residue both cross-correlated relaxation rates point to a considerably larger X angle than found in
the reference structure.
=
4
4
3
3
2
C8
4-:
-
N- 2
rI 1
'r
N- 1
-0
rDC8A
5(
8-
^
CWHl:Nl
gi
°
11'
-1
------ : "V'
I
J
4-
-t 3
-; -
/
11
0
*
-
-
'S
\
r-'°°'GSA
rC2'H2'N1
-3
-3-
0
90
180
270
x[o]
Figurw
19 Fr csAi(sd
iu)
ixd
Itre&abe 'rf. Te *em
0
90
180
270
360
% [0]
andr
Pcssie zk x1 'wifor dx nmrd
isMaid '.
360
rDDCSA
C2'H2',Nl
: 06V__97'e
I
-A
rDDC&A
C'lNl
inXam
2CN
DA
(das
hed
)fr
wh am sun by I&& dws.
bet'wnd t crs-canan
i z a* Wmuba
t& im oys-"dawd ?daa
70
qthe14-ntRNA.
U7( )adrig)
exars
rateag
a shz by dd&ed
ratesaregimv
Chapter 2: F(HCN)-Experiment
2.4 CONCLUSIONS
In this part of my thesis, I have introduced two new NMR-experiments aiming at the
determination of the glycosidic bond angle X in oligonucleotides from the angular dependence of CG
H-dipolar, N CSA cross-correlatedrelaxationrates (r'jc4
1
and rD4f
s -,,csl/9
'
HICI,NI/ ,
-",
a2nd2, 619
Nl,9)
A /8
This comprised the development of a new pulse sequence as well as the critical examination of the
data obtained from the experiments.
2.4.1 Pulse Sequence Development
The pulse sequences developed here for the determination of
DLoCSA,
all rely on a quantitative
approach. This method is advantageous to a J-coupled one in that the latter requires the evolution of
coupling, which increases the number of signals in the spectrum and reduces the sensitivity both by a
factor of 2. In general, however, the quantitative method requires a more elaborate pulse sequence in
order to suppress unwanted relaxationpathways and transfer the cross and the reference operator as
similarly as possible. In addition, while for the J-coupled approach either only one spectrum is
necessary or both required spectra are obtained at the same time, the spectra in the quantitative
approach are run in general consecutively due to the inherent different intensity of cross and
reference signals, thus requiring higher stability of the experimental setup. Here, a quantitative
approach was chosen, since the aim was to develop a method for the application to larger RNA
molecules, where resolution as well as sensitivity become the limiting factors. During the course of
my work on this project, an alternative method for the determination of dipole-dipole, CSA cross
correlation in an HCN spinsystem was presented [27], relying on a J-coupled approach, which was
applied to a 37-nt RNA. This work, however, focused on the analysis of the different factors
contributing to dipole-dipole, CSA cross-correlated relaxation in regard to anisotropic overall and
internal motion and the contributions of
D ,CSA
rHC'
cN
rFI
CSAand
rDDcsA
No attempt was made to use
as a structural parameter for the determination of X.
The quantitative
(HCN) developed in this report has been optimized
enhancement during the long periods, in which
a significant sensitivity improvement
3C
for the TROSY
is transversal. While this optimization constitutes
for the base moiety because of the large CSA of the aromatic
carbons, it has been shown that for the ribose moiety HCN-correlations can benefit from the use of
71
Chapter2: F(HC)-Experiment
CH multiple quantum coherence (MQ) more than from the TROSY enhancement connected to C
single quantum coherence [14, 28]. In principle, the introduction of MQ constitutes only minor
changes in the pulse sequence scheme and could prove advantageous especially for larger molecules
with unfavorable relaxation behavior. Since sufficient sensitivity could be obtained in reasonable
experimental times (about a day), no optimization in this direction has been carried out.
2.4.2 Experimental Quality
As shown by the application to two RNA molecules of known structure, X can be determined
from r4'
sI
HI1',N 9
alone as well as from the ratio of
C'
1/9
and F-basewith a precision of about
10° . This accuracy is equal to the one of the X angles resulting from the analysis of 3J(C,)
coupling
constants, which is the standard method for the determination of this angle to date. The precision is
similar for the 14-nt and the 30-nt RNA, demonstrating that the underlying parameterization is valid
in regard to the neglect of anisotropic overall motion and the use of
13 C
and
1 5N
(CS-tensors from
mononucleoside crystals in the solid state. The agreement between the measured and the reference
angles, however, is considerably better in canonical stem-regions than in the loop regions. This is
surprising, considering that the agreement of experimental and theoretical F-base values is in general
worse in the canonical regions as compared to the loop regions. This discrepancy could be due to the
dependence of
H'C
4
dependence on the
1 1 9,?
13 C
on the 15N CS-tensor alone, whereas F-base exhibits an additional
CS-tensor. It follows that the agreement between the experimental and
15 N CS-tensor.
theoretical F-basevalues might not represent a measure for the correctness of the
separate values of the
1 5N
and the
13 C
The
CS-tensors and their conformational dependence of both
tensors can unfortunately not be further analyzed based on this data. It is possible that while the
reference
15N
CS-tensors are applicable to oligonucleotides in solution, the
The comparable quality of the fit to the reference values of
z '
FHDI
s
13C
CS-tensors are not.
angles determined from
/ 9 alone as compared to those derived from the F-rato, however, contradicts this
assumption. To date, only one complete study of
(mononucleoside
13C
and
15N
CS-tensors in model compounds
crystals) is available [8]. All of these model compounds
are in the anti
conformation. With regards to a possible dependence of the CS-tensor on conformation, F(x)
parameterizations will clearly benefit from a direct, residue specific determination of the nitrogen and
72
Chapter2: F(HC()-Experiment
carbon (CS-tensor in the bases of oligonucleotides in solution. In principle, the fully anisotropic "5N
CS-tensor can be determined from the geometric information conveyed in multiple cross-correlated
relaxation rates correlating the N CS-tensor to the different neighboring dipole-dipole vectors or
from the anisotropic chemical shift information extracted from samples in different alignment media.
For proteins, a number of studies have been carried out along these lines aiming at the determination
of the amide ' 5N CS-tensor in solution [29-31]. Although these methods assume a number of
simplifications such as the axial symmetry of the CS-tensor or a common tensor value for all sites,
they could be expanded to the site specific determination of the fully anisotropic CS-tensor of N9
and N1 in oligonucleotides.
Since both
angle determination methods from the F(HCN)-experiment
can claim the same
accuracy, the decision as to which of the methods is applied depends only on the time considerations
and whether or not the system under investigationis dynamicallycharacterizedby NMR.
Apart from knowledge about the
15
N CS-tensor, the angular interpretation of F1 1 DCSA -rates relies
on information about the global and local dynamics of each molecule under investigation. Here, the
accuracy of the method has been investigated by employing almost entirely rigid model molecules
(only one loop residue is mobile for both molecules). In the case of conformational averaging,
however, X cannot be extracted from the F(HCN)-experiment without detailed dynamic information.
This requires the separate measurement of autocorrelated relaxation rates and a model free analysis
of this data. Although
this dynamic characterization
is part of every extended
investigation, in cases where this information is not available, the ratio of
r?
4,,
9
molecular
and F-basecan
be used for angle determination. Although this method is more time consuming, it offers the same
precision than rDcs a9
alone, whereas the parameterization requires no adaptation to either
overall correlation time or magnetic field strength. However, it has to be noted that while formation
of the F-ratioeliminates the dependence on concerted motions of the entire nucleotide, the effect of
different motions in the base as compared to the ribose moiety, like e. g. rotations around X or ribose
repuckering, is not removed and will impair angle determination even from the F-ratio. In addition, it
has to be kept in mind that the same concerted motion can affect the two GH dipole-dipolevectors
in different ways, leading again to a difference in the two F-rates. In case of a more complicated
dynamic behavior the F(HCN)-experiment can be complemented by 3J(C,H) coupling constants.
73
Chapter 2: F(HCN)-Experiment
The
angles obtained from the g(HCN-experiment
applied to the ribose moiety have been
incorporated directly into the structure calculation of the 30-nt RNA in addition to a set of easily
obtainable nOe distances. Inclusion of the additional restraints resulted in a significant improvement
in the RMSD of the resulting structures, signifying that the direct %angle restraint can replace a more
time consuming nOe analysis. The direct incorporation of the angle restraint is, however, only
preliminary. In the future, the measured F-rate needs to be incorporated together with the F(X)
parametenrizations.
The F(H'C2'N)-experiment was intended to complement the F(HCN)-experiment and remove its
inherent ambiguity in angle determination. Derivation of the F(X) parameterization showed that only
in the case of non-canonical C2-endobut not for canonical Cy-ezdo sugar-puckering
I",c2sAN/
9
offers an independent parameter for the determination of X. Investigation of the F(H2'C2Nexperiment applied to the two residues in the 14-nt RNA, which adopt C2,-endoconformation did not
result a significant improvement in angle determination. In addition, X angle determination from the
experiment is dependent on a detailed characterization of the ribose pucker mode. A separate
parameterization is required for each ribose pucker. Therefore, instead of using the F(H2'C2'N)experiment, ambiguous structural information from the F-(HCN-experiment can be complemented
by nOe or scalar coupling constant information.
Since the overall RMSD in case of the more anisotropic 30-nt RNA is of the same order of
magnitude as the RMSD of the 14-nt RNA, we conclude that the effect of diffusion anisotropy is not
significant and can be safely neglected in the size range of up to at least 30 nucleotides.
Due to the linear dependence of cross-correlated relaxation rates on both the magnetic field
strength and the overall correlation time t,
the F(HCN)-experiment
represents an interesting
method for application to larger systems. It has been successfully applied to a sizeable RNA of 30
nucleotides without severe loss in sensitivity, implying that the loss in signal intensity due to faster
relaxation of the larger RNA is sufficiently counteracted by the linear increase in the crosscorrelation rate.
74
Chapter 2: F(HCN)-Experiment
2.5 REFERENCES
1.
Singer (1984). Principles of Nucleic Acid Structure. New York Springer Verlag.
2.
Ban, N.; Nissen, P.; Hansen, J.; Moore, P. B.; Steitz, T. A. (2000). The complete atomic
structure of the large ribosomal subunit at 2.4 A resolution. Scoe, 289, 905-920.
3.
Wijmenga, S.; Mooren, M. M. W.; Hilbers, C W. (1995) NMR of Macromolecules. Oxford
University Press Inc.: New York p. 217-288.
4.
Schwalbe, HI; Marino, J. P.; King, G. G; Wechselberger, R; Bermel, W.; Griesinger, C
(1994). Determination of a complete set of coupling constants in 3 Glabeled
oligonucleotides.J Biord NMR, 4, 631-644.
5.
Trantirek, L.; Stefl, R.; Masse, J. E.; Feigon, J.; Sklenar, V. (2002). Determination of the
glycosidic torsion angles in uniformly 3GClabeled nucleic acids from vicinal coupling
constants 3 J(C2)/4-H1'and 3 J(C6)/8-Hl'. J Bicn NMR, 23, 1-12.
6.
Munzarova, M. L. and Sklenar, V. (2003).DFT analysis of NMR scalar interactions across
the glycosidicbond in DNA. J AmOJienSoc, 125,3649-3658.
7.
Schwalbe, H; Carlomagno, T.; Hennig, M.; Junker, J.; Reif, B.; Richter, C; Griesinger, C
(2001). Cross-correlated relaxation for measurement of angles between tensorial interactions.
Metdxs E rdnzyn,
338, 35-81.
8.
Stueber, D. and Grant, D. M. (2002). (13)C and (15)N chemical shift tensors in adenosine,
guanosine dihydrate, 2'-deoxythymidine, and cytidine.JAmChemSoc, 124, 10539-10551.
9.
Allain, F. H. and Varani, G. (1995). Structure of the P1 helix from group I self-splicing
introns. JMd Bid, 250, 333-353.
10.
Ennifar, E.; Nkulin,
A.; Tishchenko,
S.; Serganov, A.; Nevskaya, N.; Garber, M;
Ehresmann, B.; Ehresmann, C; Nikonov, S.; Dumas, P. (2000). The crystal structure of
UUCG tetraloop.JMol Bid, 304, 35-42.
11.
Ohlenschlager, O.; Wohnert, J.; Bucci, E.; Seitz, S.; Hafner, S.; Ramachandran, R.; Zell, R.;
Gorlach, M (2004). The Structure of the Stemloop D Subdomain of CoxsackievirusB3
Cloverleaf RNA and Its Interaction with the Proteinase 3C Stuaue (Ginh), 12, 237-248.
12.
Stoldt, M.; Wohnert, J.; Gorlach, M.; Brown, L. R. (1998). The NMR structure of Escherichia
coli ribosomal protein L25 shows homology to general stress proteins and glutaminyl-tRNA
synthetases. Eni7oJ, 17, 6377-6384.
13.
Guntert, P.; Mumenthaler, C; Wuthrich, K. (1997). Torsion angle dynamics for NMR
structure calculationwith the new program DYANA. Mol Bid, 273, 283-298.
14.
Fiala, R; Czemek, J.; Sklenar, V. (2000). Transverse relaxation optimized triple-resonance
NMR experiments for nucleic acids.JBio'd NMR, 16,291-302.
15.
Sklenar, V.; Peterson, R. D.; Rejante, M. R.; Feigon, J. (1993). Two- and three-dimensional
HCN experiments for correlating base and sugar resonances in 15N,13Clabeled RNA
oligonucleotides.J Bind NMR, 3, 721-727.
75
Chapter 2: F(HCN)-Experiment
16.
Farmer, B. T., 2nd; Muller, L.; Nikonowicz, E. P.; Pardi, A. (1994). Unambiguous throughbond sugar-to-base correlations for purines in 3 C, 5N-labeled nucleic acids: the
HsCsNb,HsCs(N)bCb, and HbNbCb experiments.JBi
NMR, 4, 129-133.
17.
Marino, J. P.; Diener, J. L.; Moore, P. B.; Griesinger, C. (1997). Multiple- Quantum
Coherence Dramatically Enhances the Sensitivity of CH and CH2 Correlations in Uniformly
13C labeled RNA. JAm U(nSc, 119,7361-7366.
18.
Sklenar, V.; Dieckmann, T.; Butcher, S. E.; Feigon, J. (1998). Optimization of tripleresonance HCN experiments for application to larger RNA oligonucleotides. J Magn Resan,
130, 119-124.
19.
Brutscher, B. and Simorre, J. P. (2001). Transverse relaxation optimized HCN experiment for
nucleic acids: combining the advantages of TROSY and MQ spin evolution. J Biond NMR,
21, 367-372.
20.
Felli, I. C; Richter, C; Griesinger, C; Schwalbe, H (1999). Determination of RNA Sugar
Pucker Mode from Cross-Correlated Relaxation in Solution NMR Spectroscopy. JAm (Cnem
Soc, 121, 1956-1957.
21.
Emsley, L. and Bodenhausen, G. (1992). Optimization of shaped selective pulses for NMR
using a quatemrniondescription of their overall propagators. J. Magn Reon, 97, 135-148.
22.
Geen, H and Freeman, R (1991). Band-Selective Radiofrequency Pulses. Janml of Matic
Rsonane, 93, 93-141.
23.
Marion, D.; Ikura, M.; Tschudin, R.; Bax, A. (1989). Rapid recording of 2D NMR-spectra
without phase cycling - application to the study of hydrogen-exchange in proteins. J Magn
Reson, 85, 393-399.
24.
Shaka, A. J.; Barker, P. B.; Freeman, R. (1985). Computer-Optimized Decoupling Scheme for
Wideband Applications and Low-Level Operation. Jaml qfMagncResonane, 64, 547-552.
25.
Riek, R. (2001). Characterization of hydrogen bond lengths in Watson-Crick base pairs by
cross-correlated relaxation. JMagnReson, 149, 149-153.
26.
Dajaegere, A. P. and Case, D. A. (1998). Density Functional Study of Ribose and
Deoxyribose Chemical Shifts. Jamml fPsica1 ChmistryA, 102, 5280-5289.
27.
Ravindranathan, S.; Kim, C H; Bodenhausen, G. (2003). Cross correlations between "3 C'H
dipolar interactions and 15N chemical shift anisotropy in nucleic acids. J Bior NMR, 27,
365-375.
28.
Grzesiek, S. and Bax, A. (1995). Spin-locked multiple quantum coherence for signal
enhancement in heteronuclear multidimensional NMR experiments. J Biorrd NMR, 6, 335339.
29.
Tjandra, N.; Szabo, A.; Bax, A. (1996). Protein Backbone Dynamics and
1 5N
Chemical Shift
Anisotropy from Quantitative Measurement of Relaxation Interference Effects. J A m Cben
Soc, 118, 6986-6991.
30.
Fushman, D. and Cowburn, D. (1998). Model-Independnet Analysis of
15N
Chemical Shift
Anisotropy from NMR Relaxation Data. Ubiquitin as a Test Example. J A m Cern Soc, 120,
7109-7110.
76
Chapter 2: F(HCN)-Experiment
31.
Boyd, J. and Redfield, C. (1999). Characterization of 1 5N Chemical Shift Anisotropy from
Orientation-Dependent Changes to 5N Chemical Shifts in Dilute Bicelle Solutions. J Am
CemSoc, 121, 7441-7442.
77
Chapter 2: F(HCN)-Experiment
78
Chapter 3: RNA Tetraloop Dynamics
CHAPTER 3
Dynamic Investigation of RNA tetraloops by
NMR
79
Chapter 3: RNA Tetraloop Dynamics
Specific Aims
The aim of this project was to contribute to the understanding of oligonucleotide stability and
dynamics on a molecular level utilizing NMR spectroscopy. Autocorrelated relaxation measurements
were used to report on the dynamic behavior of multiple carbon sites within every residue of two
members of the same tetraloop family, the exceptionally stable cUUCGg tetraloop and the less stable
uCACGg loop [1, 2]. The NMR relaxation data were translated into motional parameters by
application of a model-free analysis and appropriate motional models, thus providing a highly
resolved dynamic picture of both loop motifs in their ground state as well as at the initiation of
melting of the uCACGg loop.
TABLE OF CONTENTS
3.1
INTRODUCTION ..................................................................................................
3.1.2
82
The tetraloop RNA secondary structure motif ......................................................................
82
3.1.2.1
The YNMG motif .............................................................................................................
82
3.1.2.2
The uCACGg motif ..........................................................................................................
84
3.1.3 Dynamic investigations on RNA byNMR ............................................................................
85
3.1.4 Autocorrelated relaxation rates ................................................................................................
87
3.1.5 Model-free formalism ................................................................................................................
89
3.1.6 Motional models .........................................................................................................................
92
3.1.6.1
Wobble-in-a-cone Model (WIA )Q
..................................................................................
92
3.1.6.2
Gaussian Fluctuation Model (GAF)...............................................................................93
3.2
MATERIALS AND METHODS.............................................................................
3.2.2
94
NMR spectroscopy ....................................................................................................................
94
1
3.2.2.1
H, 5N-HSQC spectra .......................................................................................................
13
3.2.2.2
C relaxation parameters .................................................................................................
3
3.2.2.3
J(H,.,H 2) coupling constants ..........................................................................................
3.2.3
Relaxation data analysis .............................................................................................................
94
94
95
96
3.2.3.1
Extraction of the relaxation data ....................................................................................
96
3.2.3.2
Model-free
3.2.3.3
3.2.3.4
Diffusion model ................................................................................................................
97
Rotational correlation time ..........................................................................................
98
analysis ...........................................................................................................
96
3.3 RES ...........................................................................................
ULTS
99
3.3.2
Temperature dependence of the imino proton resonances .
13
................................................
99
3.3.3
C relaxation analysis .............................................................................................................
13
3.3.3.1
C relaxation parameters ..............................................................................................
3.3.3.2
Model-free analysis ........................................................................................................
3.3.3.3
101
101
107
Dynamics of the 14-ntRNA ........................................................................................
115
80
---
Chapter 3: RNA Tetraloop Dynamics
3.3.3.4
3.3.3.5
3.4
Dynamics of the 30-nt RNA .............................................................................
119
M otional implications....................................................................................................123
DISCUSSION .........................................................................................................
131
3.4.2 M odel-free analysis..................................................................................................................
131
3.4.3 Dynamics of the YNM G tetraloop motif ...........................................................................134
3.4.3.1
Ground state dynamics .................................................................................................
135
3.4.3.2
Melting of the uCA CGg motif.....................................................................................136
3.4.3.3
Biological implications...................................................................................................
138
3.5
OUTLOOK .............................................................................................................
139
3.6
REFERENCES .......................................................................................................
140
81
Chapter 3: RNA Tetraloop Dynamics
3.1 INTRODUCTION
3.1.2 The tetraloop RNA secondary structure motif
Loops are stretches of 3 to 8 nucleotides connecting an intramolecular base paired region, thus
allowing for a reversal of the strand direction, which is necessary for double strand formation. They
are part of all larger structured RNA molecules. Tetraloops are the most common loop structures in
ribosomal and messenger RNA [2]. They constitute key sites for folding nucleation [3, 4], tertiary
structure formation [5, 6] and interactions with proteins [7-9]. These functions seem to require a
well-defined
structure,
resulting
in an
in uw
preference
for
sequences,
which
adopt
thermodynamically stable secondary structures. The most common tetraloop sequence motifs are
GNRA, UNCG and CUUG (N=any nucleotide; R=purine; Y=pyrimidine), which constitute over
70% of the tetraloop structures found n iw [10]. The members of these families each share a
common fold. In contrast to the less stable GNRA family, UNCG tetraloops have not been found
involved in tertiary structure formation and also do only rarely take part in RNA-protein interactions
[11, 12].
3.1.2.1 The YNMG motif
Recently, the UNCG motif has been absorbed into the more general YNMG (M=A or C)
tetraloop family [12]. The UNCG subfamily has been consistently structurally characterized both by
NMR-spectroscopy [13, 14] and X-ray crystallography[15-17]. The structural features of the UNCG
motif are shown schematically as well as within the structure of a cUUCGg-loop in Figure 1 and can
be summarized as follows: Residues L1 and L4 form a base pair with L4 in the sn conformation.
The base pair is stabilized by a bifrucated hydrogen bond (interactions a and b in Figure 1) and a
hydrogen bond between the 2'-hydroxyl group of L1 and the L4(0 6 ) (c). The actual loop is formed
by L2 and L3, which both exhibit C,-edo ribose puckering, thus extending the backbone by about
2A [18] and bridging the two sides of the stern. The base of L3 forms a hydrogen bond with the
backbone phosphate group of L2 (d). The conformation of this residue is further stabilized by an
intraresidual base-to-ribose hydrogen bond (e) and stacking interactions with the base of L1 (grey
circles in Figure 1A).
82
Chapter 3: RNA Tetraloop Dynamics
B
19
.4
S
5'
3'
5'
3'
cUUCGgl [15] (B).
Figul 1: Samc sedary s
e of the UNCG tetra. nuif (4 and siu ef
ad
lis
(ader
Odnn bnzk) and grey
boz),
Stabiizg ieractan are gen as dashd lims (WatsoaCrideban
dades (ase sta&ir.
4onm
b
The
lo
Ribose rtis
in the C2'-endo
nmt
are soi
in gre the grey stred base is in a syn
residesare lab"d L1 to L4; the es f the doing bse pair am la
iweaaios acrgiig
to thestu awf
S and S2. The Hbdgm
thetetralosare (a) L4(N)-L 1(O); (b) L4(N)-L1(Q); (c) L1(02 )-
L4(4; (4 L3(N4)-L2(PR); (e)L3(C)-L3(0);(~ L2(~-).L4(0.
While residue L2 is stabilized by a hydrogen bond between its 2'-hydroxyl group and the base of
L4 () in a UUCG-loop X-ray structure [15], no such interaction was found in the NMR [14] and
both base and ribose moiety of L2 are free from any interaction with the other residues in the loop.
In the X-ray structure, base stacking interactions are found between L2 and L1 and from L1 across
the strand onto the closing base pair.
From the NMR-structure as well as from a closer inspection of the X-ray structure, intrastrand
stacking of L1 onto the closing base pair, as shown in Figure 1A, seems more likely. Structural
investigations at low detail indicate that other members of the YNMG family exhibit structural
characteristics similar to the ones of the UNCG motif [12]. In one particular case, the uCACGg
motif, a detailed NMR structure [1] demonstrates a virtuallyidentical structure to the cUUCGg loop
(Figure 2).
In defiance of the structural similarities between the cUUCGg and the uCACGg, the
thermodynamic stability of the latter is significantly lower [1]. While the cUUCGg loop is the most
stable know tetraloop motif (TM=72 .9°C) [12, 13], the uCACGg loop melts already at around 45°C
[1].
83
Chapter 3: RNA Tetraloop Dynamics
L2
Il
itionq tdx cUUC-g[15] (dk gy) andd uCA
d Mg nz4[1] (izgt gy).
Figut 2 S : pn
The decreased stability of the uCACGg loop has been attributed to its U:G wobble closing base
pair, which supposedly has a destabilizing effect in contrast to the GG base pair of the cUUCG
loop.
The general relevance of the closing base pair for loop stability is supported by studies showing
that tetraloop stability is decreased upon reversal of the CG closing base pair to a G:C base pair [19].
This has been explained by the different propensity of interstrand base stacking interactions between
the first loop residue and the closing base pair.
3.1.2.2 The uGACGg motif
The 30-nt uCACGg hairpin RNA studied in here constitutes a part of the regulatory cloverleaf
structure in the 5'-non-translated region of the coxsackievirus 3B (CVB3) genomic RNA (Figure 3).
The loop is part of the stem-loop D (SLD), which constitutes the major recognition site for the
viral protein 3CDP ° [1, 20, 21], the precursor of the protease 3
'
0.°
and the polymerase 3Dp
Interaction of 3CDP ° with SLD is an essential step in the assembly of the viral replication process
[22, 23]. In utro interaction studies have shown that SLD alone is sufficient for binding of 3CDPr° .
Replacement of the uCACGg by other members of the YNMG family (cUGCGg) does not impair
the interaction with 3CDP
°
[1, 24], indicating a structure based rather than a sequence based
recognition mechanism.
84
Chapter 3: RNA Tetraloop Dynamics
A
i t-
Translation Start
I
5' -NTR
Coding Region
B
SLD
SLA
5'
Figure 3:A' Shmuc
(NTR) is iicat
uralprn
3'
"pesenan f theacsadk iem B3
the 5'-dowdafisbhigbigba B: C
i
0?
hesxnay smuae
dafsewSary smcw and nn
f the5'-non-trarlsad
rgm
aw
The bidng site f the
3CD- is viSa!t
3.1.3 Dynamic investigations on RNA by NMR
Autocorrelated relaxation parameters constitute a measure of the rate of decay of non-equilibrium
magnetization through different mechanisms. Since NMR-active nuclei can be viewed as tiny
magnets, their motion will result in magnetic field fluctuations, which cause nearby nuclei to relax.
The rate of relaxation therefore constitutes a measure for a nucleus dynamics. The contribution by
other spins to the relaxation behavior of a nucleus depends on the internuclear distance as well as
their magnetic susceptibility. Therefore, e. g. a protein backbone amide nitrogen experiences
primarily the relaxation effect due to the proton directly attached to it. Hence, the analysis of the
15N
relaxation data will report on the dynamics of the amide N-H vector. The quantitative dynamic
analysis of NMR relaxation data is most simply and therefore most widely applied to systems, which
can be treated as isolated I-S spin systems. In this, I represents a hydrogen spin and S is the
85
Chapter 3: RNA Tetraloop Dynamics
heteronucleus ( 3 C
15,
whose dynamics is studied. It is assumed that the heteronucleus relaxes
predominantly due to the I-S dipolar interaction and its own CSA.
In proteins, the amide nitrogen in
15N
only labeled molecules represents a residue specific dynamic
probe (all amino acids can be sampled except proline, which does not possess an amide proton)
exhibiting to first approximation the relaxation behavior of an isolated I-S spin system (Figure 4,
left). In contrast, in oligonucleotides, due to the faster imino as compared to amide hydrogen
exchange, NH moieties are only present in base paired regions and in most cases, only one of the
bases in a base-pair can be sampled (Figure 4, right).
Protein
I --..A
,..o--
RNA
P-1
N
WI
I
1./V
k
- I&-
-A.
Figure 4:NH-tars asdyancpri
inprmei (10 and rmeicaids (nigbt.
In order to obtain more detailed dynamic information on RNA,
15N relaxation
measurements have
to be complemented by the investigationof other nuclei. 13C relaxationcan report on both the ribose
and the base moieties of all nucleotides. It has, however, to be noted that unless selective labeling
schemes are employed, C in the bases of guanosine and adenosine and C2 in adenosine represent the
only isolated CH groups in nucleic acids. For the remaining
S spin system relaxation but the effect of neighboring
3C
13C
sites, 13C relaxation does not follow I-
nuclei (CCdipolar contribution) has to be
taken into account in a quantitative analysis. In addition, the chemical shift (CS) tensor of both the
aromatic and the aliphatic carbons is not axially symmetric [25, 26], which further complicates the
analysis.
Because of the afore-mentioned
complications, oligonucleotide
C relaxation data has been
obtained and analyzed by resorting to selective labeling techniques in order to create isolated I-S spin
systems, like selective labeling of specific "3Csites [27-31], uniform partial
86
13C enrichment
[32, 33] or
Chapter 3: RNA Tetraloop Dynamics
measurement at
13C
natural abundance [34-36]. If data was obtained on uniformly labeled samples,
the analysis was either restricted to the isolated C2 and C8 spins in adenosine and guanosine [37],
small systems, in which the CCdipolar contribution is small compared to the CH contribution [38,
39], the data was only qualitatively analyzed [40-42] or a larger number of relaxation parameters has
been acquired [42].
In order to investigate the molecular basis for the higher stabilityof the cUUCGg compared to the
uCACGg motif, the 14-nt hairpin RNA comprising a cUUCGg tetraloop and the 30-nt SLD,
containing the uCACGg motif have been studied by relaxation measurements at 298K, where both
loops are stable and at 317K, where the uCACGg loop has started to melt, while the structure of the
cUUCGg loop is unperturbed. At both temperatures R,, Rp and hetnOe measurements have been
carried out for C1 and CQin pyrimidines and C1 . and C8 in purines (Figure 5). In order to obtain
quantitative dynamic information, the relaxation data was subjected to a model-free analysis and
translated into motional models. In addition, the ' 5 N line-width was studied at both temperatures.
A
NH2
I
I
N
H
OH
Figure 5: Imesgtd
1
Cs (liZitgey)inps
(4) andpyqiid
OH
(B).
3.1.4 Autocorrelated relaxation rates
Relaxation of a spin S in an I-S spin-system is dominated by the I-S dipolar interaction and the S
chemical shift anisotropy (CSA). In case of a
13 C spin
with an attached proton, the spin-lattice (R1 ),
the spin-spin (R) and the steady state nOe (hetnOe) relaxation parameters are described by the
following (Eq. (1)-(3)) [43]:
87
Chapter 3: RNA Tetraloop Dynamics
R 1 =d
(1)
4
[J(oH - CO) + 3J(coc ) + 6J(COH+ OC)]+ C2 J(cOC )
R2 =d8 [4J(O)+J(H - c)+3 J(coc)+6J(co
H ) +6J(CO
H +CO)]
(2)
2
+
HetnOe=l+(d 2 /4R,
(3)
In this,
6 [4J(O) + 3 J(coC )]+ Rex
0
H
/c
X6J(cH +C )-J(H % -C
)]
and c0 c are the Lamor frequencies of H and 1 3 C, respectively, J(co) is the spectral density
function at frequency
a, Rex is the parameter accounting for the conformational exchange
contributions; d = uohycyH /(8 rCHr
2
) and c = COCAcc //[
describe the dipolar and the CSA
interaction, respectively, uo is the permeability of the vacuum; h is Planck's constant;
is the
gyromagnetic ratio of nucleus i; r, is the internuclear distance between C and H; Ac c is the carbon
CSA
The principle dependence of the relaxation parameters on the overall rotational correlation time tc
is shown in Figure 6. While R1 exhibits a maximum, R2 increases continuously with
c. This
difference arises from the dependence of R2 (Eq. (2)) but not of R, (Eq. (1)) on a J(O) term, which is
negligible at lower tc values but becomes more and more relaxation relevant with increasing tc. For
macromolecules such as proteins or RNA, Tcusually lies right of the R, maximum and consequently
small R1 but large R2values can be expected.
88
Chapter 3: RNA Tetraloop Dynamics
.--
100
C
I
;... .; :-.: .,.
C.;.,
. , :.-- - .:-''.:.':'.:'':''
U)~~~~~~~
7'
~~'10
-in 0~~~~~h',"¥""'-
___ __
':"'"'""' it! .'.'"'.', v'ai
.-- :..:-.Pne
rO
w
0
I
.
,
2
.
I
4
.
6
I
8
w
I
10
'r [ns]
Figuwe 6:Depen
H
torl
fR 1, R 2 and thehet
on r . A rmngeticfield
smh
lf1.09A hs been
assund Thercngi f themIas
qf 600MHz, a CSA of 60ppmanda C
stuiedn e has
ir
at bygreysd
3.1.5 Model-free formalism
The model-free approach [44, 45] has been developed in order to translate autocorrelated
relaxation rates into general dynamic parameters, which are free of any underlying motional model,
hence 'model-free'. This is desirable, since NMR relaxation data unfortunately does not contain any
information on the nature of the motions causing the relaxation. The model-free formalism is the
most widely applied method for the dynamic interpretation of relaxation data. Spin relaxation is
considered simplified within an isolated I-S spin system, e.g. the relaxation mechanisms are restricted
to the I-S dipolar and the S CSA relaxation. Its output are the so-called model-free parameters.
Different models of varying complexity have been developed to describe the amplitudes and time
scales of motion (Figure 7):
In the simplest case, these consist of the generalized order parameter S2 , which is a measure for the
spatial restriction of the internal motion, and the effective correlation time Tedefining the time scale
of motion (Figure 7).
Assuming isotropic diffusion, the spectral density function can then be
expressed as follows (Eq. (4)):
(4)
J(co) = 2
S22
+( _
o892
89
)r
Chapter 3: RNA Tetraloop Dynamics
In this, c is the overall rotational correlation time of the molecule and 1/c=l/'Cc+l/c
e.
If
'Ct
is
small (tc < lOps),the dynamics can be described exclusively by S2 (Model 1, Figure 7A).
In the presence of slow motional events in the milli- and microsecond range Models 1 and 2 can be
extended by a chemical exchange or line-broadening term Rex (Figure 7C&D, Model 3& 4). In
principle, the Re-term accounts for the sensitivity to R2 for slow time scale motions. In the modelfree analysis, R= is directly added (see Eq. (2)). Direct interpretation of R= is however difficult since
the requirement for this term can arise not only from its own conformational exchange but also
other sources like chemical exchange of adjacent parts of the molecule.
In cases, in which two fast motions with time scales differing by at least one order of magnitude
take place, an extended model-free formalism has been developed [46] with a separate S2 for each
motion, S 2 and S2 (Figure 7E, Model 5).
(5)
J(o)=2
(1-S 2 )rf
S 2rc
L+crL
1+2
C
+ o 2T
2
~f
(S-S2)T
+ co2
J
In this two time-scale model (Eq. (5)), it is assumed that the contribution of the faster of the two
motions can be neglected (f+O0). Therefore, while the faster motion contributes to the overall S2
(S2=S
2
*S
), the term containing the fast effective correlation time f is left out. Similar to
simpler models, the time scale of the slower internal motion ;s is included in
will therefore not be discriminated from
Cein
(1/T =l/
Tc
in the
+l/t1) and
the following.
In the case of an anisotropic shape, the molecule does not longer tumble with one isotropic
rotational correlation time (Figure 7F). For the case that D =D,7 =D1 , the spectral density function
of a symmetric top rotator (D =D I ) or an axially symmetric top molecule is obtained (Eq. (6)):
(6)
Jq( ) I(jq, +jq,L
J+q,mean
,in which the reduced spectral density functions is:
(87)
~jq,m
2rm
+9(0qm)
90
------
Chapter 3: RNA Tetraloop Dynamics
A
C
B
R.
o-22
s2
IN
..
E
D
'
.
.
.-..
:
F
R-,
Figur Z7:
Midd-fiwrparant fr a GH spasstem thna n e thaturns icirad y th de oerall aal
a vatir t
c(A-E) or amsfally y (l. A Maidel
1;Fast er mo onetrn sale ( < lOps;S241). B:
Modd 2; Skver ire
mtian (r,> 0lps).C Mdd 3 Exend Modd 1 rwgi
te dxnical exdmr xwa
(rf< lOps;S2 41, R A2).D:Modd 4; Ext d M
0). E:Moadl 5; Thotnm scaleextended
rndd
40, r ; 0) F A nisp
axiallysynt
zt M
dion
D Il and D ae the g and the short axis f te ax
We2, oorping the
S2
paranmt,
In tbisa
y syn
S and S
heoa
nda
nt/ibution(rc> 1Ops,RE •
forslowandfastnii
tul
ling
d/insm teor,
arc7wd the short and the long axis of the rraule and rcm ean is the aeragd
91
enicalexdnge
2
is desti
<;
by nation
rzI and r aye
ti
(S
te
at/ntis
f
s;
Chapter 3: RNA Tetraloop Dynamics
In Eq. (7), m={ Il,
I
, man}. The correlation times
can be related to the diffusion tensor
components Di1 and DL according to Eq. (8):
II-,m = D i +n'(DI, -D I
(8)
with n=O for m= L, n=1 for m= j and n=2 for m=nran
3.1.6 Motional models
Although the advantage of the model-free analysis is that a specific motional model is assumed a
prio, in the end, a motional picture from the dynamic analysis is desirable. It has, however, to be
kept in mind that the relaxation data do not report on the nature of the motion but only on its
extent. Therefore, any motional model has to be examined critically, since it might origin more in our
desire to condense the data into a picture than it reflects the actual processes in the molecule.
Two slightly different models for the motional interpretation of S2 are presented in the following.
3.1.6.1
Wobble-in-a-cone Model (WIAC)
NH2
H
OH
Figure 8: 'WaWu na-ame-nrddfor Cr-Hr arndC8-H8in ader
The so-called 'Wobble-in-a-cone' (WIAC) model [47] allows modeling of the motions of the CH
vector as motions on a cone of semiangle Oo.(Figure 8) with the time scale
order parameter as follows (Eq. (9)):
S 2 =[1/2cos0 0 (1+cos9 0 )]2
(9)
92
~ ~~~~~~~ ~ ~ ~ ~ ~ ~ ~
------
ce.
00 is related to the
Chapter 3: RNA Tetraloop Dynamics
3.1.6.2 Gaussian Fluctuation Model (GAF)
.0
X
IH2
N
2
L.
H
OH
OH
Figur 9: GAF-nllfor
Ct118amEd X in adep
The GAF model [48] represents a restricted version of the Wobble-in-a-cone model since it
describes a Gaussian distribution of bond vectors on the surface of a cone (Figure 9). The S2
parameter and the angle between the CH vector and the cone-axis is defined as (Eq. (10)):
(10)
S 2 =1 3 sin2
cos2 l
- e- X
)
-sin2
4
( 1
-e
4
)l
In this, fi is the angle between the C-H vector and N-Ca,, the director axis of the cone, and a
2
is
the standard deviation of the fluctuation in the azimuthal angle, which corresponds to the fluctuation
inmx.
93
Chapter 3: RNA Tetraloop Dynamics
3.2 MATERIALS AND METHODS
3.2.2 NMR spectroscopy
The NMR samples of the 14-nt and the 30-nt RNA were prepared as described in the Material and
Methods section of Chapter 2. In case of the 30-nt RNA, the relaxation data was obtained from two
samples. One, with selective
3 C and ' 5N
labeling in the guanine and cytidine residues was obtained as
described as described in Chapter 2. The second exhibits selective labeling in the adenine and uridine
residues and has been obtained with a similar synthesis as well as sample preparation scheme. All
measurements
1H{13 C/15 N}
were carried out on a 600MHz Bruker spectrometer
equipped with a 5mm
Z-Grad TXI probe at 298K and 317K. The spectra were processed using Bruker
NMRSuite (XwinNMR 3.5) programs.
3.2.2.1
H,SN-HSQC spectra
'HK 5N-HSQC spectra were obtained with the nitrogen carrier set to 154ppm, the spectral width in
the indirect dimension was 20ppm and 64 complex points were acquired in the indirect dimension
with 4 transients per t increment. The proton carrier was at 4.7ppm with a spectral width of 25ppm
and complex 1024 points were acquired. For the 14-nt RNA, in addition to measurements at 298K
and 317K, a spectrum was obtained at 323K.
3.2.2.2 uC relaxationparameters
R1, Rip and heteronuclear nOe data were obtained for C1 , C6 and C8 from 13C modifications
(Appendix A3.1) of methods introduced for
1 5N
autocorrelated relaxation measurements [49-52].
Separate experiments were conducted for C1, and the aromatic carbons with the carbon carrier
frequency set to 89ppm and 139ppm, respectively. The spectral width was 6 ppm for Cr and 5 ppm
for C6/C. 64 to 100 complex points were acquired in the indirect dimension depending on the
available spectrometer time. The proton carrier was set to the water frequency (4.7ppm), the spectral
width was 10ppm and 1024 complex points were acquired in this dimension. Off-resonant carbon
Q3 pulses (512s) were applied during carbon evolution with an offset of -7500 Hz or-5000 Hz in
order to suppress the J(C,C 2 ) and lJ(Cs,C) coupling, respectively. R1 and Rp data was obtained
with 8 scans, the hetnOe with 16 scans for each t,-increment. The 2D-R- and Rlp-subspectra with
94
Chapter 3: RNA Tetraloop Dynamics
varying relaxation delays were acquired in interleaved pseudo-3D
experiments
were also recorded
experiments. The hetnOe
interleaved, with alternating proton-presaturated
and non-
presaturated transients. The interleaved spectra were separated by a slightly modified Bruker standard
macro (splitinvnoe, Appendix 3).
For the acquisition of R1 relaxation rates, spectra with a varying relaxation delay TM of lOmns,
50mnis,
100ms, 200ms, 400ms, 700ms, s and 1.5s. Spectra with rM=50ms and 400ms were recorded twice.
Rp rates were acquired as described in ref. [51] and [52]. Random length proton decoupling pulses
were applied during the carbon spin-lock period (option d in ref. [511). Adiabatic Mulder pulses [52]
were used to rotate carbon magnetization into the transversal plane. A spin-lock field of 3.6 kiHz was
applied for the variable rM delay (M=12ms, 24ms, 36ms, 48ms, 64ms, 8ms,
104ms, 128ms).
Duplicate measurements were carried out for 24ms and 80ms. The spin-lock offset was set to
2000Hz.
Th
e
2000HIz.
The measured
Rameas-rates contain a spin-lock offset and spin-lock power dependent
contribution of R. From Ras
(11)
and Ri, R2can be extracted followingEq. (11):
R2=
R meas- R1 cos2Os
2
sin 2 Os
In this, 9, is the angle of the effective spin-lock field for each nucleus with the B0 field. 9, is defined
according to Eq. (12):
(12)
Os =tan-(
s
,in which v is the spin-lock field strength in Hertz and .2s is the resonance offset from the spin-
lock carrier in Hertz.
3.2.2.3
3 J(Hr,H .)
2
3 J(H .,H ,)
1
2
coupling constants
were obtained from E.COSY splittings of H1 ,G2 and H 2,C, cross-peaks in a 2D-
HCCH-E.COSY experiment [53]. The carbon carrier frequency was set to 77ppm with a spectral
width of 40ppm and 128 complex points in the indirect dimension. 64 transients were acquired for
each t increment. The proton carrier was positioned on the water resonance at 4.7ppm. 1024
95
Chapter 3: RNA Tetraloop Dynamics
complex points were acquired with a spectral width of 10ppm. Errors were obtained from duplicate
measurements.
3.2.3 Relaxation data analysis
3.2.3.1 Extraction of the relaxation data
From the spectra, peak intensities were obtained using Felix2000 (msi) macros (autopkhpos.mac,
Appendix 3.2). The hetnOe constitutes the ratio between the peak intensities in the spectrum in the
presence and in the absence of proton presaturation. The error was obtained from duplicate
measurements. In cases, where no duplicate measurement has been carried out, an error of 1% (0.01)
has been assumed.
For both R1 and R1 p, mono-exponentional 2-parameter decay curves were fit to the peak intensity
versus relaxation
delay correlations
using the
macros
compkhuncledit2.s,
myfx2gnu2p.s,
myrun.r2.2ps and mycurvefit2table provided by Palmer and co-workers [54]. The error on the data
incorporated into the fitting procedure can be calculated bythe macro compkhuncledit2.s from the
different peak intensities obtained in the duplicate measurements of two time points. This errors,
however, frequently proved to be too small for the subsequent fitting routine to produce relaxation
rates within the a =0.05 critical value. Therefore, the error, which was defined differently for each
time point by the program, was increased to the maximum calculated error for each time point. In
some cases, even this error did not suffice. In this cases, the error was increased in small steps until
all residues could be fit. It has to be pointed out, that this procedure did not have any effect on the
resulting relaxation rates but did only change the resulting uncertainty in the values.
3.2.3.2 Model-free analysis
The model-free analysis of the relaxation parameters has been carried out using the program
Modelfree 4.15 by Arthur G. Palmer [55]. Modelfree is able to fit the relaxation data with the 5
different dynamical models (Figure 7).
While initial runs were performed separately for CQ and C/C4, final Modelfree runs were carried
out with the combined relaxation data.
During model selection the number of models accessible for a given residue is defined by the
number of relaxation parameters obtained for this residue, since the number of fitted parameters
96
Chapter 3: RNA Tetraloop Dynamics
must
not
exceed the
experimental
parameters.
Model
selection
followed the
procedure
recommended in ref. [55] and is briefly described in the following:
Occams razor was applied in that the simplest model fulfilling the required level of certainty was
selected, even if a more complicated model improved the fit. Accordingly, the selection process was
started applying Model 1 to all residues. The goodness-of-fit
between the model and the
experimental data was determined by creating 200 randomly distributed synthetic data sets using
extensive Monte Carlo numerical simulations. These data sets represent the random variations in the
fitted data arising from the error of the experimental data. The results were used to determine the
cumulative probability distribution and the a =0.1 critical value of the sum-squared error
X2.
Model
1
was selected, if the x 2 of the residues was below the a =0.1 critical value. For the remaining residues,
model 1 was compared to either of the two-parameter models using an F-test analysis, which is
included in Modelfree. If the 0.2 critical value of the F-test analysis was exceeded, the more
complicated model was selected. If the data could not be fitted with either of the two-parameter
models and the at=0.1 critical value for Model 1 was not significantly exceeded, the data was still
fitted with Model 1. For the remaining residues, the three-parameter models (Model 4 and 5) were
applied and compared to Model 1 accordingly. If the 0.2 critical value of the F-test anaylsis was
exceeded, the respective model was selected, otherwise, the residues were considered not fittable.
The x2 values are dependent on the varying error resulting from the preceeding curve fitting
procedure for R, and Rip. In the model-free analysis, a general error of 5% has therefore been
assumed for R1 and Rp. This error was considered appropriate to account for the possible error in
the assumed CSA values together with deviations in the pdb-coordinate
file from the actual
structure.
In case of the hetnOe, the error resulting from duplicate measurements sometimes amounted to
considerably less than 1%. In order to fit the data in the model-free analysis, the error was elevated
to 1%. A lower error was applied for the hetnOe than for R1 and R2 since its dependence on
anisotropic diffusion is not as pronounced.
3.2.3.3 Diffusion model
The diffusion model was selected by fitting the relaxation data to an axially symmetric or an
isotropic diffusion tensor in the program Modelfree. In addition, the diffusion tensors has been
determined from hydrodynamic calculations using the program hydroNMR [56] based on the pdbcoordinate files of the molecules.
97
Chapter 3: RNA Tetraloop Dynamics
3.2.3.4 Rotational correlationtime c
The rotational correlation time c was determined by different methods. On the one hand, c was
determined from the
13C
relaxation data using the program Modelfree, where it constitutes a part of
the diffusion model optimization. On the other hand, it was taken from the output of the program
hydroNMR, which performs a hydrodynamic calculation based on a pdb-coordinate file. In addition,
a rough estimate of c can be obtained from a simplification of Eq. (4). In the absence of internal
motion (S2 -1 and -rz--), J(co) simplifies to Eq. (13):
J(§) =
rc
+co2 r2
,and Tccan be determined from the R2 (Eq. (2)) over R 1 (Eq. (1)) ratio according to Eq. (14):
(14)
J
2
R.
2 2
98
Chapter 3: RNA Tetraloop Dynamics
3.3 RESULTS
3.3.2 Temperature dependence of the imino proton resonances
The exchangeable imino protons constitute detectors for the presence of hydrogen bonds in
nucleic acids. If an imino proton is detectable, it is engaged in a hydrogen bond and the peak linewidth is an indicator for the strength of the interaction in respect to its structural as well as its
dynamic features. If the imino proton is not part of a hydrogen bond, it exchanges with the solvent
water and is therefore not detectable. Figure 10 shows the 1H, 15N-HSQC spectra of the 14-nt and the
30-nt RNA at 298K and 317K. In the following, relative resonance intensities have been used as
measures for the line-width. Since the integral of a resonance is invariant, the intensity reflects the
line-width in a comparative analysis. All imino resonances of the 14-nt cUUCGg RNA stem residues
including the closing base pair are of similar intensity at room temperature.
A
U7 C8
U .............
oil
ppm
6
144-
CS--G:
317
G9
146-
A4-Ull
3
_Gt0
148-
C - G12
150-
G2-C13
152-
*G12
G2
154-
G5' 3'-C14
5'
3'
156158160162-
ul.
.
14.0
.
13.5
.
13.0
1 2.
12.5
1 2.
12.0
1 ..
11.5
1
11.0
1.p0. .
10.5
1 .0
10.0
ppm
B
0 5 el6
na
_
C14-- Oj
G1t' - PP"
c" .......
UlJ- G1
8
144
uCG!,
A tL - 'If'
0
U11t- A2
G l_ C 21
G0u8
C 2 223 15
U 2 4 154
U"
GU25 154
G.
C7
U s -C
A 4 -U 2 7
C3- G28
G2_ U2 '
G 1 - C3 0
5'
4
15
3'
154
16(
16
13.5
13.0
12.5
12.0
11.5
11.0
10.5
10.0
ppm
n
Figuw 10: lHl 5 N-HSQCspmra jhe 14-nt(A and de 30-nt RNA (B) at 298K(to) and 317K (boari. ID sli:e
grey).
dmogh he
resonanmnv
of
bt the basepar in ds
an the tw a4amm basepans am gw (shaded nm
99
Chapter 3: RNA Tetraloop Dynamics
The G9 imino resonance is only slightly smaller in intensity, signifying that the U6:G9s base pair
is about as stable as the Watson-Crick base pairs in the sternm.
At 317K, the G9 imino resonance is significantly decreased, indicating that the U6:G9s base pair
is still present but considerably weakened. All the other base pairs, even the loop closing base pair,
remain stable. The G9 iino
resonance is still present at temperatures as high as 323K (data not
shown).
In case of the 30-nt uCACGg RNA, the iino
resonance of the loop base pair C14:G17
is
undetectable at 317K and the resonance of the closing base pair U13:G18 as well as the one of U19
adjacent to it are weakened.
This relative decrease in signal intensity at similar positions in the hairpin demonstrates the higher
stability of the cUUCGg as compared to the uCACGg loop. Observation of the imino resonances
alone, however, does only report a limited number of residues, especially since three residues of both
loops do not show an imino resonance in the spectrum. In addition, imino resonances undergo
exchange reactions that complicate the analysis of their relaxation behavior.
100
.
Chapter 3: RNA Tetraloop Dynamics
3.3.3 3Crelaxation analysis
3.3.3.1
3Crelaxation parameters
R 1, R2 and hetnOe data of C1, and C6 or C of all residues in the 14-nt and the 30-nt RNA at 298K
and 317K are sunmmarized in Table 1 to Table 4. Typical R 1 and R2 relaxation delay curves for the
14-nt RNA are shown in Figure 11.
RI [s' ]
G1
G2
C3
A4
C5
U6
U7
C8
G9
G10
Ull
G12
C13
C14
C 6 /C 8
3.54+0.04
3.62+0.06
4.43+0.07
3.68+0.04
4.60+0.10
4.53+0.06
3.49+0.04
4.08+0.06
3.30+0.03
3.69+0.04
4.68+0.07
3.71+0.04
4.48+0.09
4.45+0.08
R2 [s4]
C1
2.50+0.16
2.68+0.04
2.76+0.03
2.81+0.04
2.85+0.04
2.89+0.04
2.59+0.06
2.54+0.08
2.62+0.04
2.65+0.04
2.75+0.03
2.77+0.04
2.86+0.04
2.79+0.04
C 6 /C 8
9.42+0.57
9.30+0.97
13.39+1.22
10.35+0.63
13.05+1.80
13.25+1.16
12.65+0.66
14.22+1.20
11.75+0.50
10.01+0.55
14.02+1.24
8.75+0.69
13.77+1.59
13.23+1.45
C 6 /C 8
C1
9.32+0.57
1.12+0.03
1.12+0.03
10.24+0.71
8.42+0.47
1.10+0.01
8.48±+0.62 1.14+0.001
8.39+0.51
1.10+0.01
8.47+0.58
1.10+0.02
8.33+1.00
1.16+0.03
1.10+0.02
6.13+1.00
9.84+0.68
1.12+0.01
8.35+0.57
1.13+0.02
8.88+0.50
1.09+0.02
9.55+0.60
1.10+0.01
7.83+0.49
1.10+0.003
1.13+0.01
6.78_+0.45
Cq,.
1.21+0.00
1.14+0.02
1.16+0.02
1.18+0.002
1.15+0.02
1.14+0.002
1.12+0.09
1.11+0.05
1.15+0.003
1.12+0.05
1.15+0.01
1.13+0.03
1.18+0.03
1.22+0.04
t e 14.ntRNA at 298K
svndaton
rates Cf/C8 and Cvqb
Table l:A u
R2 [s 4 ]
R1 [s"]
G1
G2
C3
A4
C5
U6
U7
C8
G9
G10
Ull
G12
C13
C14
HetnOe
HetnOe
c 6 /C 8
CT
C6 /C 8
C,
3.78+0.09
3.92+0.09
4.69+0.12
4.11_+0.10
4.99+0.14
5.08+0.13
3.62+0.08
4.54+0.11
3.57+0.07
3.94+0.08
5.08+0.14
3.99+0.08
4.88+0.13
4.92_0.13
3.20+0.09
3.38 +0.03
3.51+0.04
3.53+0.04
3.57+0.04
3.51±0.04
3.23+0.05
3.32+0.07
3.30+0.04
3.34+0.04
3.52+0.04
3.46+0.03
3.53+0.04
3.23+0.03
7.03+1.49
11.51+3.14
9.52+2.29
7.37+1.72
9.09+2.68
9.26+2.37
9.31+0.94
9.93+1.59
8.99+0.97
7.45+1.54
9.71+2.41
10.30+2.86
9.58+2.36
9.33+2.01
7.73 +0.40
6.50+0.36
6.33+0.37
6.26+0.35
6.54+0.41
5.15+0.53
5.73+0.77
7.34+0.44
9.53+0.63
6.48+0.36
6.57+0.32
6.19+0.37
5.06+0.28
Table 2:Autama
natan
C6 /C8
1.22+0.01
1.18 0.002
1.13+0.002
1.16_+0.01
1.20+0.007
1.15+0.01
1.21+0.002
1.16+0.003
1.19+0.001
1.19+0.001
1.15+0.01
1.18+0.009
1.15+0.007
1.15+0.002
C1
1.23+0.02
1.18+0.01
1.17+0.01
1.21+0.00
1.21+0.01
1.18+0.01
1.23+0.02
1.22+0.02
1.21+0.01
1.24+0.30
1.21+0.02
1.21+0.03
1.19+0.02
1.32+0.01
ratesofQC/C8andC, fte 14-rtRNA at 317K
101
Chapter 3: RNA Tetraloop Dynamics
G1
G2
C3
A4
C5
U6
C7
U8
G9
G10
Ull
A12
U13
C14
A15
C16
G17
G18
U19
A20
C21
C22
HetnOe
R2 [s ]
RI [s]
C1 ,
C1
C 6 /C 8
C 6 /C 8
1.70+0.03
1.64+0.03
1.91+0.04
1.58_+0.003
1.94+0.04
.
.
2.04+0.03
2.02+0.002
1.59+0.02
1.59+0.02
2.06+0.004
1.63+0.001
2.00+0.002
1.98+0.03
1.49+0.001
1.90+0.03
1.53+0.09
1.65+0.02
2.00+0.003
1.64+0.003
1.89±0.04
1.99+0.04
C1 .
1.06+0.03
1.11+0.01
1.18_+0.01
1.07+0.02
1.12+0.02
c 6 /c 8
26.53+1.41
25.02+0.93
37.19+2.15
23.01+1.01
35.54+2.42
1.04_+0.02
1.10+0.02
1.01+0.02
1.15+0.02
1.11+0.02
1.13+0.02
1.15+0.03
1.27+0.02
1.38+0.05
1.30+0.02
1.40+0.01
1.25±0.02
1.22+0.02
1.06+0.02
1.20+0.01
1.12+0.01
36.36+2.03
32.04+0.43
24.74+0.97
24.89+0.90
32.27+0.75
24.22+0.40
28.72+0.33
36.58+1.99
16.37+0.13
33.71_±1.42
31.31+4.64
23.92+0.70
29.27+0.15
23.83+0.39
36.35+2.24
37.33+2.13
1.11+0.01
30.50+2.35
1.08+0.01
24.14+1.07
1.08+0.02
38.81+4.99
1.10+0.01
29.71+2.01
1.09+0.01
25.25+1.07
1.07+0.01
22.96+1.14
25.50+1.44 1.10+0.003
1.12+0.02
28.44+1.90
15.35+2.26 1.26+0.002
1.14+0.01
22.19+1.70
24.45_+1.18 1.06+0.01
1.13+0.01
25.50+1.30 1.06+0.004
1.12+0.004
24.03+1.28
25.20+1.60 1.09+0.003
1.07+0.01
27.43+1.84
1.96+0.002
1.96+0.001
1.55 +0.02
1.94+0.004
1.59+0.02
2.03+0.001
1.89+0.03
1.17+0.03
.
1.28 +0.04
31.52+0.30
30.90+0.09
26.46 +1.06
33.66+0.38
23.69+0.94
28.45+0.46
37.37+2.02
21.81+1.22
.
-
28.92+1.59
27.21+1.56
25.40+1.41
-
1.16_+0.004
1.14+0.02
1.07+0.001
1.10_+0.01
1.09+0.004
1.17+0.01*
1.15±0.01*
1.11+0.01*
1.12_+0.01
1.08+0.01*
1.10+0.01*
1.08+0.001
1.12+0.01*
1.07+0.01*
1.11+0.003
1.17+0.01
1.15+0.03
1.12+0.01*
1.23+0.01
1.18_+0.01*
1.14+0.01*
1.14+0.0l*
1.14+0.01
1.05+0.01
1.10+0.012*
1.08+0.01*
U23
U24
U25
G26
U27
G28
U29
C30
Table 3: A ut=a=ea
gaen,
-
1.07+0.01
1.25+0.02
1.18+0.03
1.14+0.002
1.10+0.000
1.11+0.01*
1.05+0.01
1.10+0.01
1.12+0.01
1.13+0.01
1.14+0.01*
1.10+0.001
1.24+0.01*
-
-
dacatn ratz CfQ/Cs and C, ef the 30-ntRNA at 298K Resick, for uoib no
d not be amazd due to ronane ozerap. In arses,
heeK (i'e
-
29.05+2.19
26.10+1.09
21.41+1.79
1.08+0.01
1.08+0.002
ere
no dupicate rmswent
byanasensk), anenvr cf1% (0.01) hasben assuwid
102
dts ae
has beencaied out for thex
Chapter 3: RNA Tetraloop Dynamics
R 2 [s]
R1 [s']
G1
G2
C3
A4
C5
c/c8
2.07+0.10
2.24+0.06
2.84+0.10
2.24+0.03
2.89+0.18
U6
C7
U8
G9
G10
UlI
A12
U13
C14
A15
C16
G17
G18
U19
A20
C21
C22
U23
U24
I,
CC
1.61+0.02
1.59+0.01
1.62+0.01
1.50+0.01
1.52+0.01
6 /C 8
HetnOe
C1 ,
I
14.98+2.94
17.95+2.89
17.78+3.14
16.20+0.81
19.84+6.73
15.07+0.85 1.20+0.003
15.30+0.48
1.16+0.01
15.38+0.49
1.08+0.01
15.99+0.36
1.10+0.02
16.15_+0.65 1.11+0.03
1.17+0.01*
1.15+0.01*
1.11+0.01*
1.14+0.004
1.08+0.01*
15.17+0.61
15.06+0.33
1.11+0.02
1.08+0.01
-
-
1.58+0.01
1.57+0.01
1.60+0.01
1.56+0.01
18.07+3.05
20.31+1.35
19.73+4.60
21.50+7.32
20.94+1.69
15.74+0.69
20.52+0.80
18.27+3.46
10.29+0.37
17.05_+2.25
15.10+6.09
12.06+1.94
19.72+1.66
14.93+0.81
19.60+4.10
20.22+3.52
1.1+0.01*
1.17+0.01
1.12+0.01*
1.07+0.01*
1.13+0.003
1.21+0.002
1.17+0.01
1.12+0.01*
1.23+0.01
1.18+0.01*
1.14+0.01*
1.14+0.01*
1.1+0.001
1.07+0.004
1.1+0.01*
1.08+0.01*
1.67+0.01
20.31+1.26
14.05+0.35
..
2.66+0.09
2.85+0.04
2.20+0.08
2.40+0.11
2.80+0.05
2.27+0.02
-
2.87+0.12
1.92+0.02
2.78+0.08
2.24+0.23
2.38+0.07
2.84+0.05
2.33_+0.03
2.74+0.12
2.82+0.11
.
.
2.81_+0.04
1.57+0.01
1.55+0.01
1.56+0.01
1.63+0.01
1.61+0.01
1.66+0.01
1.74+0.01
1.87+0.03
1.76+0.02
1.91+0.01
-
15.39+0.58
1.10+0.01
15.79+0.37
1.09+0.01
15.20_+0.37 1.12+0.01
15.23+0.37 1.16+0.003
15.27+0.54 1.12+0.001
11.33_+0.78 1.22+0.01
14.25+1.18 1.11+0.004
18.07+0.61
1.16+0.17
1.17+0.01
15.89+0.39 1.12+0.002
14.90+0.38 111+0.002
13.69+0.54 1.13+0.002
15.82+0.57
1.08+0.01
1.12+0.01
U25
G26
U27_
G28
U29
C30
C1 ,
G6 /C 8
1.2+0.004
_.
2.35 +0.09
2.30_+0.07
2.85+0.04
2.66+0.10
-
1.51+0.01
1.54+0.01
1.51+0.02
14.67+4.05
-
20.00+4.70 15.66+0.59
1.09+0.02
19.81+1.54 16.75±+0.45 1.10+0.004
20.58_+3.10 13.00+0.94
1.10+0.03
1.11+0.01*
-.
1.14+0.01*
1.14+0.004
1.24+0.012
Table 4:A torvrai daxai ratesf C/C andC, ofe 30-ntRNA at 317K Reidus,for ubh n rnemts are
g wm ld n beandyeziddw to wmame
o*dap. In ases, teren dicate rnasurent
h ben aied aztfor the
betne (iri'amd byan astesk), ane r'rof1% (0.01) hasbeenass
103
i
Chapter 3: RNA Tetraloop Dynamics
I
Pe
I
Kip
I
"7
0
..
C
_C
!
I
0
1
0.5
[
0
1.5
I
0.1
0.05
0.15
TM [s]
[
U7, G9 and G12 qe 14-ntRNA
rdxazdan
deatyaaus fC and Qfr res
11:R (ft)
and R1p(nright)
dday . Theaus epet nmapwid
at 298K The le heits (in arbary wits) areplot agmrt tenxati
Figur
2-paranrzerfits. Tk dqlite
in de gra&.
data sets, eh hw ben acq
pak
in Oierto otain emm fordie
mties, are ilm
Figure 12 shows the relaxation data for both C6/C and C. of the 14-nt RNA at 298K. These data
demonstrates two general characteristics of the measured relaxation rates:
- R1 as well as R2 relaxation rates of C6of both cytidine and uridine residues are systematically
higher than C8 relaxation rates in guanine and adenine. On average, R1 is 20%, R 2 30%
higher for the C6.
- R and R2 relaxation rates of C1. are smaller than aromatic carbon relaxation rates. On
average, they amount to only 63% of the respective C6 values.
A possible explanation for the increased relaxation rates of C6 in pyrimidines could be the presence
of a second dipole, C, in the vicinity, while C8 in purines represents a more isolated I-S spin system.
However, as discussed in detail below, the effect of the CCdipolar relaxation is too small to account
for the observed -30% difference in relaxation rates between C6and C.
An alternative explanation could be found in the difference in CSA values of C6 and C. Since the
CSA constitutes one of the two main sources of nuclear relaxation, differences in its magnitude are
likely to be relaxation relevant.
104
-
-
-
Chapter 3: RNA Tetraloop Dynamics
A
B
1 29
6
.'4
1
lb
6
3 47
12
.
m
4I
12 35
9 1
2
-
Il
W
0
20
20
I
8 13
4
.to
2
2
;
20
'15
15
10
n 5
iiii
0
0
1.5
1.5
01
D
0.5
0
04
12
9 012358131467
Al
G
C
1
0oc 0.5
I0 0
X
4
I1
C8
C6
0 12 3 5 8 13
C
- I
,I~~~----
1 2 9
A I1
U
G
_
l
.
U
_
I
C 1.
Figure 12:MeasuredR,R2 ardtnC
6Q/Cs
(4 and Ci' (B) f de 14-ntRNA stad y hase Reside mins
aregin dandabneo degraPfs.Thelop riess arelabe indrk
e o es e daing basepairin lit gry
Two studies on
13C
CS-tensors in nucleotides are available to date, one based on 13Cchemical sift
components determined by solid-state NMR experiments in combination with density functional
theory (DF)
calculations [26] and one exclusively relying on DFT calculations [25].
While these two studies agree to a varying degree for the different nucleotides (Table 5), the most
remarkable difference is that while in ref. [25] similar CSA values are proposed for C of the two
purines as well as for C6 of the two pyrimidines, ref. [26] agrees on the similarity of the purine CSA
values but reports a CSA for cytidine, which is about 40ppm lower than the one for uridine.
.
CSA [ppm]
Adenine
Guanine
C6 Cyddine
Uudine
Table 5: CSA
r. [26
ref[25]
131.4
139.6
156.8
195.5
122.8
119.0
183.9
184.1
esforC6ad CGami
toFiala etaL [25andStuer
105
etaL [26].
Chapter 3: RNA Tetraloop Dynamics
As shown in Figure 13, R1 and R2 increase by about 30% for a molecule the size of the 14-nt RNA,
when the CSA is increased from 120 to 185ppm, signifying that this difference in the CSA can
indeed fully explain the observed difference between C6 - and CQ-relaxation rates. Furthermore, the
similarity in the observed relaxation rates for cytosine and uridine residues indicates that the (CSA
values determined in ref. [25] reflect the CSA in oligonucleotides more closely than the ones from
ref. [26] and should therefore be preferablyused in the further analysis.
From Figure 13, it becomes also clear that errors in the absolute CSA are more dramatic for larger
CSA values, hence more crucial for C6 and C than for q.
C1 The CSA of C1 is about 45ppm in C
-
erdo conformation according to ref. [25] and 30ppm for Cq-erdo and 60ppm for Q,-edo as reported
in ref. [57].
A
B
7
70
6
60
5
50
X
O: 3
:
40
30
2
20
1
10
0
0
0
2
4
6
8
10
0
2
4
t, [ns]
6
8
10
T= [ns]
C
z
00C
4- 1.5
I
0
2
4
6
8
10
· [ns]
Figure 13: 3C CSA aWtibthon to 13C awda
daxation R1, R 2 and theheC haze beencadatd peig on
r, cth an assurd CSA of Oppt 30Cp 60ppn 120ppmand 185ppmat a field strth of 600 MHz. The GH
int
ar wtor nth ws set to 1.09A. The aation ti
f the 14-nt and the30-nt RNA at 298Kare iit
106
Chapter 3: RNA Tetraloop Dynamics
Even if a CSA of 60ppm is accounted, R and R2 of C1, arrive at only 68% of the respective values
calculated with a CSA of 185ppm, which corresponds to the observed differences between C1 , and
C6 relaxation rates. Hence, the lower CSA of the aliphatic as compared to the aromatic carbons fully
accounts for the smaller R 1 and R2values of C 1, compared to C6 and C.
3.3.3.2
Model-free analysis
In order to assess the validity of the desired residue specific dynamics obtained from the modelfree analysis, the global parameters extracted in the process, the diffusion tensor and
Tc,
have been
compared to those derived by independent means. Furthermore, as demonstrated above, the
important external parameter for the analysis of "3 C relaxation data is the
two available sets of
13 C CSA
3C
CSA. Therefore, the
values from ref. [25] and [26] have been compared in their potency to
produce a sensible output of the model-free analysis. Only after these methodological investigations,
the actual dynamic interpretation has been carried out.
The CCGdipolarcontribution to C6 and C1 relaxation - The model-free formalism has been
developed for an isolated I-S spin system. While it can be readily applied to investigate C in purines,
C6 in pyrimidines and C1, both possess a neighboring S-spin (C or C2.), which contributes to
relaxation through a CCdipolar mechanism The contribution of an additional CCdipole relativeto
the dipolar relaxation caused bythe directly bound proton is expressed in Eq. (15) [58]:
3 rC 1
~(1)S~ ~PHC
Pcc
crcH I°c
in which Pic is the dipolar contribution to the relaxation of the 13C nucleus arising from the
attached
H or "3 C nucleus; i is the gyromagnetic ratio of nucleus i; r is the distance between the
spins i and j; ac is the carbon Lamor frequency and T%is the overall rotational correlation time of the
molecule. The relative CCdipolar contribution of C6 and C2. to the relaxation of Co and
respectively, is depicted in Figure 14 in dependence of the overall rotational correlation time
107
Tc.
C1.,
Chapter 3: RNA Tetraloop Dynamics
4
C5 -C6
3
I0
a
2
-
C.-C 2
I
Q
1
0
0
2
6
4
8
10
TC [ns]
Figuw 14:CDian
the oerd
all
ae CGCidar
aw to CH-dar mhanism to erdaxcation
qrfC6and Cl in deewe
nt qr the intse
wndation tm t as dzoaidl inEq. (15). The stmtural arra
ad
f
dxeac4amnt
ti
frtfhe 14-rtand 303ntRNA at 298Kae irkitL A field
carbonspim(gey dis) is sho iinc)tdiG C
sttp_
f 600MHz has en assut1 The irenmdrdistn fir GH, C1-C2' and C6-Cshawe
bn set to 1.09a,
1.53A ard1.37A as traatdfrnINSIGHTII(nim.
In addition to the dependence on the nuclear magnetic susceptibilityand the sixth power of the
internuclear distance, the GCdipolar contribution increases with the square of both the correlation
time and the magnetic field strength, signifying that the CGdipolar contribution becomes more
notable for larger molecules as well as at higher fields.
Due to the dependence on the gyromagnetic ratio as well as the sixth power of the internuclear
vector, the CCq-dipolar contribution to C6 relaxationis only about 1% of the CH-dipolar relaxation
for a molecule of the size of the 30-nt RNA (=5.65ns
at 298K) and even smaller for the 14-nt
RNA (c=1.92ns). Sincethe C.-C 2. bond vector is longerthan the C6-Qvector (1.53as compared to
1.37A),the C2 Cdipolar contribution to C, relaxation amounts to only around 0.5% for the 30-nt
RNA. Taking into account CSA relaxation, the CCdipolar contribution to overall spin relaxation is
even smaller.
Therefore, for both C1 and C 6/CG, the C dipolar contribution has been neglected in the dynamic
analysis of the molecules investigated in here and CqH1 and C6 H6 have been treated as I-S spinsystems.
108
-
-
Chapter 3: RNA Tetraloop Dynamics
The Diffusion Model - The program Modelfree [55] consistently fits the combined C1,and the
C/C
4
relaxation data of the 14-nt RNA to an axially symmetric diffusion model with its unique
principal component oriented along the helix axis and an anisotropy (D= 1/)
of 1.33 at both
temperatures (Figure 15A). The axially symmetric diffusion model constitutes the most complex
model contained in the Modelfree program. Size and orientation of the diffusion tensor determined
by Modelfree have therefore been cross-validated by hydrodynamic calculations using the program
hydroNMR [56]. The tensor determined by hydroNMR is axially symmetric with its orientation
within 5 identical to the Modelfree results with an anisotropy D=/1
1
of 1.39. Both tensors are in
agreement with an earlier dynamic study carried out on the 14-nt RNA based on
15 N relaxation
rates
[59], where an anisotropy of 1.34 has been determined.
B
A
df sideziwarndtop ew The
14-nt(4)ard e30-ntRNA (B) demiinM
Figure 15:Dfiin temorqcfdhe
dfsian tenor f the 14-ntRNA is gizeningmyin mearfranrfe30-rtRNA.
For the 30-nt RNA, a diffusion anisotropy of 1.81 is fit from the relaxation data (Figure 15B). This
is validated by the hydrodynamic calculations, which arrives at an axially symmetric tensor with an
anisotropy of 1.9. The principal tensor component is again similar within about 5° for Modelfree and
hydroNMR It has to be noted that while for both the 14-nt and the 30-nt RNA, the principal
109
Chapter 3: RNA Tetraloop Dynamics
diffusion tensor component is oriented along the helix axis, due to the longer stem of the 30-nt
RNA, they are tilted about 40 ° away from each other (Figure 15). This indicates that the C-H vector
orientation relative to the diffusion tensor is not the same in every RNA even for the vectors in the
planar base, but depend on the size of the RNA.
The effect of different diffusion models on C6 /Ca and C,1. relaxation rate analysis is shown in
Figure 16. The outcome of the model-free analysis on the 14-nt RNA with an isotropic tumbling
model is compared to the same procedure using an axially symmetric diffusion tensor. In the second
case, the relaxation rates depend on the orientation of the GH vector with respect to the diffusion
tensor. While for the relaxation properties of C6 and C8 (Figure 16A), the diffusion model has almost
no effect on the stem residues, it affects residue C8 and to a lesser extent also G9. For C1., (Figure
16B), most of the stem residues are also not sensitive to the diffusion model, only the terminal
residues 1 and 2 and the loop residue 9 show differences to the isotropic model. It is remarkable that
in all deviating cases the isotropic model results in a more flexible S2.
A
1
270
0.8
180
N
C
0.6
90
0
0.4
1 2 3 4
5 6
7 8 9 10 11 12 13 14
Residue
c1.
B
270
1
Isotropic
0.8
Anisotropic
180
ea
.
0.6
.
.
v
G G C A C U
- ,. i
I . .
1 2
Figur
16:Effects o the dfi
nGon
3 4
90
!.o'
L,,
fnA
.
.
U C GG
. .
U G C C
. . .
. . .
-
5 6 7 8 9 10 11 12 13 14
Residue
the rxed-fimarlysis. C6/C8 (4) arndC' (B) S2 parantes Al
,axation data f the 14-rt RNA at 298K
sing
an i mti:
(g'y lie) or an anis
si
axy
snD
ifrm
the
(Hk irms)
gn aaateda
dfion nmi Theark 0 Qeetuntherpectie GH wts andtheprnzipleaxis f thediwon tenoramre
yM oddfire (dashac11l).
110
Chapter 3: RNA Tetraloop Dynamics
The relative insensitivity of the stem as compared to the loop residues to the diffusion model can
be understood when considering the orientation of the respective GH vectors relative to the
diffusion tensor (see angle 0 shown in Figure 16). In stem regions of RNA molecules, the diffusion
tensor is oriented about perpendicular to the base planes, resulting in angles 0 with the principalaxis
of the diffusion tensor of around 90 ° for C--H 6 and C-H
8
vectors. Since RNA is in addition almost
axially symmetric, the orientation of the GH vectors within the base plane does not affect their
relaxation properties. Hence, the relaxation behavior of all base paired residues can be expected to be
similar. Furthermore, the relaxation properties of vectors perpendicular to the long diffusion tensor
axis is governed by rotations around the principal axis, while it is less sensitive to rotations along
both other axes. Incidentally, rotations around the principal axis occur more often, signifying that
vectors perpendicular to the long diffusion tensor axis and hence for RNA vectors in the base plane
will be less sensitive to the applied diffusion model as vectors parallel to the long axis.
In contrast to the C-H
6
and C-H48 vectors, the angles 0 between C H1 . and the diffusion tensor
axis are more diverse (Figure 16B). In line with this observation, the Cq, data alone results in a similar
diffusion tensor, while the C6 /C4 data alone fits to an isotropic diffusion model.
This analysis shows that even for a molecule as small as the 14-nt RNA with an anisotropy of 1.33,
anisotropic diffusion affects the resulting dynamic parameters, especially in the most interesting loop
region, in which the C-H vector orientations deviate from the ones in the stem region. These
deviations
are, however,
only defined, if the molecule under investigation
is structurally
characterized. It follows that while anisotropic diffusion has to be included in the dynamic analysis in
order to dissect structural and dynamic effects on the observed relaxation rates, the inclusion of a
molecular structure introduces an additional potential source of error.
The overall rotational correlation time
c - The overall rotational correlation times determined
by different means for the 14-nt RNA at 298K and 317K are given in Table 6.
rc,[ns]
298K
M aydfiwbA oNMR
C6/C
1.92
2.47
R2/R,
1.72
ModfE
b mNMR
1.31
1.62
1.89
C,
Table 6: rc w1ue frdx
317K
14-rntRNA. The zc
resdingfmheanraediR
111
2 /RI
R2/R1
1.58
1.42
ratio rfalldJiestemresid
Isgwn
Chapter 3: RNA Tetraloop Dynamics
Correlation times of 1.92ns and 1.31lns at 298K and 317K, respectively, have been obtained from
the extensive model-free analysis for the combined C 6 /CQ and C1 , relaxation data. While at 298K,
these value is slightly higher than the approximate
values determinated from the R2/R1 ratio of all
T,
C1 it is slightlylower at 317K (1.58ns for C6/C 8
the stem residues (1.72ns for Cq/C and 1.89ns for Q),
and 1.42ns for C1 ). The
T,
values from the hydrodynamic calculation are about 0.6 and 0.3ns higher
than the model-free results at 298K and 317K, respectively. In spite of these deviations, the
correlation with tT/T, the ratio of the viscosity at a given temperature and the temperature is linear
for the relaxation data derived x, values (Figure 17A).
In an earlier 15N relaxation analysis conducted on the same 14-nt RNA at 273K a c of 5.7ns has
been reported [59]. This value is in better agreement with the hydrodynamic calculation than the
13C
relaxation analysis.
A
B
6
6
5
5
4
in
4
U)
3
LI
0
19
2
3
2
1
1
0
0
0
3
4
5
6
2
T/T* 105 [gm*cm'l*sec1*Kl
7
1
0
]
1
2
3
qT/T*10 [gm*cm'l*sec'l*K1]
Figur 17:Depeder q r, on e ratioaee thezsaity fuaterand a dute
dxe13C ndSxatn data uiig Malief
30-nrtRNA (B). rchas ben deUermum
erawfrr e 14-rt(4 andt
at 298K and 317K (dark grey
the
Ised Tn nic caa
u
ing
dianods), fan "15N iaxatzondata [59] at 273K (bade dot),firm sane
pmgramAbdrNMR
(litgrey sqmmar)
andfim e separate
R2/Ri rats jCV C andCQ(Hak andagly
M I ). 7he
lio mpment the Itliarfit
to e nfxId-fie (blaxk In) and d e7)dNMR
112
data(Ivy=1)
4 de ongm
Chapter 3: RNA Tetraloop Dynamics
For the 30-nt RNA (Table 7), the c of the model-free analysis (5.65ns and 3.65ns at 298K and
317K) are about 0.65 and 0.45ns higher than the xc values of the C6/C8 R 2 /R 1 ratio and about 0.2
and 0.6ns lower than the C, R2/R1 ratio. Also for the 30-nt RNA, the dependence of the model-free
results on temperature is linear (Figure 17B).
r,[I]
298K
Mcdfm
C6/8
Qi/S
_
NMR
5.65
3.67
_'__
Table Z7:rC
317K
/R1
eRAdoNMR
4.98
6.18
3.65
2.40
R2/R
3.19
3.83
fifrthe3O-ntRNA.
Although the reason for the differences between the experimental and the theoretical c cannot be
elucidated in here, the linearity of the xcversus
T/T relationship for both the 14-nt and the 30-nt
RNA establishes confidence in the model-free relaxation data analysis.
This data demonstrates that c obtained from the R2 /R1 ratios of C1. reflect the actual values better
than the one from C/C 8 .
The C chemical shift anisotropy - Model-free analysesof the C6 /C8 relaxation data obtained
on the 14-nt RNA using either the
3C CSA values taken
from Fiala et al. [25] or from Stueber et al.
[26] (Table 5) are shown in Figure 18A. If Stueber et al. CSA values are used, the average stem S2
values are around 0.8-0.85 for all nucleotides but uridine, while S2 for the only stem uridine, U11, is
significantly higher (close to 1). In addition, U6 in the loop shows an elevated S2, suggesting that this
residue is more stable than the stem residues. For the 30-nt RNA, all stem uridines exhibit higher S2
values than the other residues, too (data not shown).
In contrast, if the (CSA values from Fiala et al. are incorporated, the uridines exhibit similar
behavior to the other nucleotides. Furthermore, the fits of the model-free parameters to the uridine
relaxation data are improved in every case. As mentioned above, the main difference between the
two sets of CSA values is that while Fiala et al. calculate similar values for C8 in both purines as well
as for C6 in the two pyrimidines, Stueber et al. report similar values for C8 but a C6 CSA value for
cytosine that is about 30ppm lower than the one for uridine.
113
Chapter 3: RNA Tetraloop Dynamics
A
C 6/C 8
I
r-'T]
Fiala
,,'"
-r.".'
0.8
.-,-"---e
*
tal.
Stueber
et al,
N
cn
0.6
stem
>
C~U
G G C A
T. '.. ' . ..
..
1 2 3 4 5 6
0.4
_.
loop
U
.. r.
7 8
stem
C
11
G. . " U
. . .. . . .
9 10 11 12 13 14
Residue
C1.
B
I
l-
.
so
0.8
'60-60
stemlopse
Nc
U)
0.6
0.4
GG CA C UUC GGUGC C
1
2
3
4
5
6
7
8
9 1011
121314
Residue
Figure18:Effecf theCSAonteS2 w
mukgfmtenrdd-friansis
f te 14-ntRNAat 298KA C/C
caladted the CSA Vduesfi*nStu r et aL[26] (gy dianmrs arndlies) andFialaetal. [25] ('ade), the3
urci reii a rd
bes. B S2 wh alt
uQ a CSA ef 30pmfor al rmsdz (gay) r a CSA
30ppmfor all ids in Cy-e3domzfomtiw (1-6, 9-14) and 60ppmfor U7 and C8 (ade), hih are in C2'-end
S2 Vue
Since the R1 and R2 relaxation rates observed in the stem uridines are simnilarto the ones of the
stem cytosine residues (see Figure 12), and there is furthermore no apparent reason for an increased
stability of the uridines, the further analysis has been carried out with the values provided by Fiala et
al.. However, for both sets of CSA values the order parameters of the cytosines are lower than the S2
of the other residues (Figure 18). This is also observed for the 30-nt RNA (see Figure 19). While this
could signify that the bases of cytosines are in general more mobile than the other nucleobases, it is
not reasonable to assume that cytosine, which is in a three hydrogen bond Watson-Crick
arrangement with guanosine would be more flexible than uridine, which forms only two hydrogen
114
Chapter 3: RNA Tetraloop Dynamics
bonds with adenine. It follows that the GSA for C6 of cytosine is most likely actually lower than it
has been reported.
Figure 18 illustrates the effect of using GSA values, which deviate from the actual CSA. If the
assumed CSA is lower than the actual one, the resulting S2 value indicates a more rigid behavior and
vice versa. The error propagated into S2 is higher for C6 and C (Figure 18A), which exhibit higher
absolute CSA values than for C1r . For C 1 , the CSA has been reported to be ribose pucker dependent
[57]. Figure 18B demonstrates the difference in S2 for CQ,using an assumed GSA of 30ppm as
compared to 60ppm for residues U7 and C8, which are in C2,-endoconformation. For the higher CSA
value, S2 decreases by about 0.03ppm in both cases, indicating a more flexible behavior. The
increased flexibility of these two residues is, however, also apparent, if a CSA of 30ppm is used.
3.3.3.3 Dynamics of the 14-nt RNA
The results of the model-free analysis of the 14-nt cUUCGg hairpin at 298K and at 317K are
summarized in Table 8 and Table 9. Some of the relaxation data could not fit satisfyingly to any of
the dynamic models (grey shaded data in Table 8 and Table 9). In these cases, the results of the
model-free analysis using the simplest model (Model 1, Figure 7A) are given.
In the following, the dynamic behavior of Cq and C8 will be considered as a dynamic reporter of
the entire base moiety, while C1., dynamics will be treated as reflecting the flexibility of the whole
ribose moiety.
At room temperature, all base relaxation rates but the one of U7 can be fit with the simplest
model, signifying the absence of slow internal motions (S 2
1) as well as slow exchange processes
(R- =0) and the presence of onlyvery fast internal motions (=t--e -0).
The relaxation behavior of the U7 base moiety is more complicated in that it exhibits millisecond
exchange (Rex=2.14s-1). At 317K, more residues show slow dynamics and 17 acquires fast time scale
motions in addition to the slow exchange process. For the ribose moiety, most residues can be fit
with the simplest dynamic model, only G1 requires a small fast time-scale motion term (Te=7 .4 2 ps) at
298K and at 317K G10 acquires a R term (Rex=2.74s-1).
115
Chapter 3: RNA Tetraloop Dynamics
Base
S
Ribose
[ps]
'rTep]|
s
|
G1
G2
C3
A4
C5
0.85+0.01
0.78±0.05
0.87+0.02
0.83+0.01
0.86+0.01
0.83+0.01
0.88±0.01
0.82±0.01
0.88+0.02
0.83+0.01
U6
0.87+0.01
0.84±001
U7
C8
0.67+0.01
2.14+0.67
| R. [s ]
[ps]
T
7.42+4.05
_
0.77+0.02
0.84+0.01
0.73+0.02
G9
0.83±0.01
G10O
0.89+0.01
Ull
0.91+0.01
G12
0.91:t0.01
C13
C14
0.86+0.02
0.83+0.01
0.85+0.02
0.80+0.01
0.81±0.01l
:
0.80+0.01
_____
- 081±0.01
..
0.82±0.01
_
T=298K; xc=1.92ns, D=-1.33
Table 8:Results f the r
l-fiearsis for C6/C8 ('Base' and C'(Rikaeq ctfe 14-ntRNA
at 298K The grey sladMdata wd not befit toanmy
dym
cnxd win the O.05to 0.00 crtical
Base
S
G1
0.77+0.02
G2
0.81+0.02
[ps]
|Te[p]|'r
Ribose
1
[ps]
s
0.83+0.02
0.86±0.01
3.77+0.58
C3
0.78+0.002
0.88±0.01
A4
0.82+0.02
0.87+0.01
C5
0.80+0.02
0.88+0.01
U6
U7
0.82+0.02
0.87±0.01
C8
0.77+0.02
0.58+0.01
| R [s']
10.16+2.66
2.16+0.49
_
0.78+0.01
0.80+0.02
G9
G10O
0.75+0.02
Ull
G12
0.83+0.02
0.83 +0.02
C13
0.80+0.02
0.87+0.01
C14
0.80+0.02
0.79±0.01
1.46+0.48
0.84+0.01
0.81+0.02
0.84+0.01
2.74 ±0.63
0.87+0.01
0.86+0.01
2.11+0.55
T=317K; TC=1.31ns; D =1.33
Table 9: Resultsof the nmxde-fiee
amys is for C6/C8 ('Base') and Cl' (Ribe)
f tde 14-nt RNA
at 317K The greyshadeddata acd not hefit to any dynmic mnIeld i te 0.05 to 0.00 acitid
udue
116
_
.
.
..
..
,
_
.
.
Chapter 3: RNA Tetraloop Dynamics
~298K
~4
I.
^V
+/
.
0.8 N
-::~...:..:.
..'~: :.-..:'."..
.
0.6
G ....
,;-; .-
..... .\
{,.4s.::.-....:..
...
Ft~~ibose
. ',,,-1'' ''.':...''' ^,'..-'.,,''-.'' ' ':.
GGC
3
4
5
,"'
'
~.' '-
C G'G
ACUU
2
1
'" ''.-
6
7
8
C2 .- endo
-10
.
...'..'...'-:,'--..- ~...
.. . C-.
;-.'.''.-....'-.
.:.;,. . ' ' .:.-.
.;...
:i ,'u ' .,:.,.:.
,~.;,"."'-....-.,
....::.:. ,.t:
:~.......
.
lA-
+~~Ba
'"
........ -.....-
-5
,
N
.. -r' ,_,
- N
Ca
-
':. ...
UG C C
9101112
O
1C
3 -endo
C3.-endo
13 14
Residue
317K
1--
Ribose
0.8 -
-
";'A
''
*
cm:
CDU.:
. \
/
Base
i
.'.''"'''''''''"'"
.:..'\i.';."!.')'-:.
"''''
"..'.:.':':-':?.
.. .................
:."
...{'
-:-'-.''
0.6
1
.
.'5
G C A C U U C G G U! ~G C~ C ~~~
I
I
I
I
I
I
I
I
1
2
3
4
5
6
7
8
I
I
9 1011
I
.
10
i:!-...............
.
'..
.. :'...--............
'~~~~~~'.'
'
. '-...,..'..'-..
:- - '
. : .:-.:.:..
'. ....'. -'b'.'--,'.:-.~.
0.4GG
C2.-endo
152
I
5
. ". -rOI'.......
~.endo
CC
3 -endo
II
121314
Residue
Figur 19:S2paramrtesfor C6/C8 ('Base', a sdid lies) and Cl (Ribse' gey solid line)f the 14-ntRNA at 298K
(ahoz) and317K( o. For thedlo and the w a4arenues,
3J(Hr,H29)clg
costnts aregienfpossible(a
dashed
la).
S2 values for the base and the ribose of the 14-nt RNA at 298K and 317K are shown in Figure 19.
From these order parameters, the following observations can be made:
At both temperatures and for the base as well as for the ribose, the stem residues except for the
terminal ones exhibit very similar dynamics. Thus, the average S2 for the bases in the stem is 0.88
117
'
Chapter 3: RNA Tetraloop Dynamics
with a deviation of only 0.003 at 298K and 0.81+0.006 at 317K. For the ribose, the stem mobility is
even more uniform with 0.83 +0.001 and 0.87 +0.001.
While the overall S2 pattern across the sequence is remarkably similar at the two different
temperatures for both the base and the ribose, the flexibility increases at higher temperature for the
base but decreases for the ribose. A possible explanation for this contrary temperature dependence
could be found in an entropy compensation mechanism. Below the melting temperature, an increase
in base flexibility cannot be compensated enthalpy-wise, but has to be compensated entropically by
decreased ribose flexibility. 3J(H11,H2.) coupling constants are in agreement with an increased rigidity
of the ribose moieties at higher temperature (Figure 19). For the loop residues U7 and U8, which are
in
2 .-ei
ribose conformation,
3 J(I-H.,H ,)
1 2
values are large. Upon temperature increase, these
coupling constants increase as well, indicating an even more rigid conformational arrangement.
In general, the similarity in the dynamic profile along the sequence at the two investigated
temperatures signifies that although the overall flexibility changes, no significant difference in the
dynamic behavior of any specific residue occurs. This confirms that the cUUCGGgloop is stable at
317K. In addition to its structure, also its flexibility does not change within the investigated
temperature range.
In particular, the dynamics of the cUUCGg motif are discussed in the following: In this, the
dynamics of the loop residues are interpreted in reference to the stably base paired and stacked stem
residues, which are considered rigid. Whereas the bases of U6 and CS are as rigid as the stem
residues and the one of G9 exhibits only slightly increased flexibility (S2 =0.83 at 298K), the base of
U7 is significantly more flexible (S2 =0.67). In addition, the ribose moieties of U7 and C8 are flexible
but to a lesser extent. These observations are in agreement with the reported structure of the
cUUCGg loop. The flexible base of U7 has been reported to be the only one, which is not stacked
but protrudes into solution. Furthermore, neither flexible ribose is involved in hydrogen bonding.
Although in general the dynamics do not change at higher temperature, the flexible base of U7
becomes even more flexible in respect to the rigid ones. The S2 of this moiety decreases from 0.67 at
298K to 0.58 at 317K. Thiis gain in flexibility is accompanied by a change in the dynamic
characteristics in that the fast internal motions show a slightly slower time scale ( < 10ps at 298K;
10ps at 317K). This is in agreement with a larger amplitude of the internal motion.
At both temperatures, the closing base pair of the cUUCGg loop exhibits dynamics similar to the
stem base pairs, indicating a rigid arrangement.
118
Chapter 3: RNA Tetraloop Dynamics
3.3.3.4 Dynamics of the 30-nt RNA
The model-free results of the 30-nt uCACGg RNA at 298K and 317K are summarized in Table 10
and Table 11, respectively.
As for the 14-nt RNA, most of the residues can be fit to the simplest dynamic model at 298K,
indicating an overall low extent of internal dynamics. The only truly flexible residue at this
temperature is A15, the second loop residue. Both the base and the ribose moiety of this residue
exhibit a complex dynamic behavior comprising fast internal motions at two different time scales.
The similarity of the spatial restrictions (S 2 =0.63 +0.02 and 0.67 +0.06 for the base and the ribose,
respectively
9 7 2 +193 ps
S
,2
=0.82+0.02 for both moieties) as well as the time scales (e=
6 07+60ps
and
for the slow motion of the base and the ribose, respectively e < 10ps for the fast motion
in both moieties)in the two moietiespoints to a concerted motional behavior of the whole residue.
At higher temperature, the base but not the ribose moiety of residue A15 experiences an increase
in flexibility. Remarkably, the model-free analysis allows to attribute the gain in flexibility exclusively
to a change in the slower motional process, which becomes less restricted (S 2 drops from 0.63 to
0.58) and slower in time scale (e increases from 607 +60ps to 750 +147ps).
Apart from A15, a number of residues, which are not flexible at lower temperature, show
dramatically increased dynamics at 317K. At this temperature, the base of A12 constitutes the most
flexible moiety of the 30-nt RNA. Interestingly, it exhibits extremely unrestricted motions (S2 =0.46)
on a very fast time scale (e<
lOps). Apart from that, this base moiety experiences millisecond
exchange processes (Rex=6.4s-). In addition to the base also the ribose of A12 becomes more flexible
at higher temperature but to a lesser extent. Albeit small in amplitude, this moiety shows motions on
two time scales. Furthermore, the base of G18 in the loop closing base pair exhibits fast dynamics on
two time scales.Although the extremelyslow time scale of the slower motion and the high error of it
(1800ps ±670), does not seem credible by itself, the relaxation data can only be fit with this model,
indicating that the presence of two motions is valid, even if the precise time scale is not.
Unfortunately, due to lacking experimental data, no information about the ribose of G18 is available.
Apart from these residues close to the apical loop, also the ribose of U25 in the distal bulge of the
30-nt RNA shows increased dynamics.
119
Chapter 3: RNA Tetraloop Dynamics
Ribose
Base
_,
~
2
S
2
Ss
222
S.
G1
0.89+0.02
G2
0.87+0.01
C3
0.81±0.02
A4
0.84±0.01
0.84±0.02
0.89+0.01
Ss2
S
Te
[ps]
|Re
[S- 1]
0.73+0.02
11.92
±4.47
8.63+1.60
0.79+0.01
0.90+0.01
6.38+1.98
C5
0.86+0.02
U6
C7
0.86+0.01
U8
0.85+0.01
0.82±0.01
G9
G10
U11
A12
U13
0.85+0.01
0.76+0.02
0.85 0.01
0.91+0.01
0.87+0.01
0.88+±0.01
0.87+0.01
0.90+0.02
0.78+0.01
3.98+1.98
0.93+0.02
0.84+0.01
C14
0.83+0.01
A15 0.51+
0.02
0.81+0.01
C16
2
S2
Rex [S]
Te[ps]
0.92+0.01
5.40+1.97
0.63+
0.02
0.82+
0.02
607.1+
59.97
0.55+
0.07
0.87+0.01
G17
G18
0.93 +0.04
0.96+0.01
0.87+0.01
0.86±0.01
U19
0.83±0.01
0.89+0.02
A20
0.86±0.01
0.78+0.02
C21
0.81+0.02
0.94+0.01
C22
U23
0.850.0l
0.89+0.01O
U24
U25
G26
U27
G28
U29
0.83o+0.01
0.89+0.02
C30
0.780.02
0.67+
0.06
0.82+
0.07
______
______
972.31+
192.59
_
0.82+0.01
0.95+0.03
0.83±0.01
0.82+0.01
0.85+0.01
0.89+0.01
0.85+0.01
0.97±_0.02
-______
0.83+0.02
-________
+
_____
___________
15.04
±2.53
T=298K; tc =5.65ns; D =1.81
Table
analysis of the 30-rnt RNA
10: Results of the nmSdel-fimee
.
aralyzeddue to reoane oear
at 298K Residues, for
ico res ults are gizen, cw
not be
Thegreyshadd data awddnot befit to any rrndl zithin the 0.05 to 0.00 critical iues.
120
Chapter 3: RNA Tetraloop Dynamics
Base
S
2
Ribose
te[PS]
2
S
PS-]
Rex[]
S2
G1
0.74+0.03
13.26+2.93
G2
0.79+0.02
6.73+3.23
C3
0.76+0.02
0.83±0.01
A4
C5
0.81+0.01
0.80
0.78+0.03
0.81±0.01
C7
U8
0.74+0.02
0.79±0.01
G9
0.79+0.03
G10
0.99+0.03
0.80±:0.01
Ull
0.81+0.01
0.85±0.01
2.36+0.85
2
Ss
S
SS 2
[p]
e[ps]
R[s]
Rex[S1 ]
0.74+0.01
2.95±0.86
0.76±0.01
2.26±0.51
±0.01
U6
0.81+0.01
6.39+0.77
6.39+0.77
A12
_= 0.46+0.01
A-2 0-46±0.01
U13
C14
0.80+0.03
16.83+5.45
0.76+0.02
0.58+
A15 0.47+
C16
G17
-
0.82±
0.02
0.01
0.01
150.84
0.72±
0.03
0.84±
0.04
823.96±
180.04
0.88±0.01
0.85±0.01
0.75+0.02
0.77+0.03
0.87±0.01
G18 G18 0.53+
0.06
0.02
0.71+
0.03
0.81+
0.05
0.74+
0.07
750.19+
147.18
0.80+0.01
0.80i±0.01
A20
0.83+0.01
0.78±0.01
C21
C22
0.78+0.02
0.83±:0.01
0.80+0.02
0.82±:0.01
U23
U24
0.81+
0.75±
0.01
0.02
U25
-
G26
0.73+0.05
U27
-
G28
0.89+0.02
0.84 ±0.01
U29
0.81+0.01l
0.82±0.01
0.78+0.02
3.60 ±0.63
1843.14+
668.08
U19
C30
0.94± 619.41+
0.77±
0.61±
0.04
0.79±:0.01
0.03
1.66±0.37
0.76±0.01
4.16+0.95
0.80±0. 0.94±0. 727.51±
01
02
210.65
51.55+22.10
__
0.72±0.01
1.6084
- ~~~~~~~~~~~~~~~~~~~~~1.60
T=317K; Tc=3.65ns;D=1.81
foruhich no dresuts
aregiwenculd notbe
Table 11:Resuts f tle rrd-fiee analysisf the 30-nt RNA at 317K Residues,
arnyzed due to resonaxeozeap. Thegey shadl data
dd rt befit toany rnrdelzuithin the 0.05 to 0.00 itical wues.
The order parameters for the base and the ribose moieties of the 30-nt RNA at the two
temperatures are depicted in Figure 20. The stem residues of both moieties exhibit in general similar
S2 values. The only exception is the base moiety of G10, which has a highly increased S2. This is,
121
Chapter 3: RNA Tetraloop Dynamics
however, not due to a bad fit in the model-free analysis since the relaxation data fits well satisfyingly
to the simple Model 1.
In contrast to the dynamics of the 14-nt RNA, the stem ribose moieties are on average slightly
more rigid than the base moieties at both temperatures. Upon temperature increase, a concerted
increase in flexibility is observed.
298K
1
~..+..i ,
r..F , :/l,,
*
0.9
_
~I~
0.8
~,, .
/.:.
. Ribose
Base
0.7
Jbulgp:
I
bulae
u!o0.6
C2 .-endo
0.5
. q:
0.4
'"'
~.
' ~'":
Yp'08-*1qs,."+;,
i :.::':;-'-,:,ii
' .:.'~'.:i. =:
..:'.- ~''r'. .'.!:.'::''.:::-'
; .:.t";' 'i ':
0.3
0.2
'
. '-,-.',' .:..-= . ';-::..:
..r.:.-':
.. ,,!:.:.': .'.:..... :' ',^,:1
.!...- .:i':::~~~~~~~~~~~~~.
'"' ':.: ..i ::':: ' ''....,.;.'.
:.i-'-::::!.'7!
1
0
,.: ''.-:':i:
' ::
?;-." ,'.!.'.';}!;
'",i~~".:i
;.:i:
GGCA
. UG
.
.
. . ..C U UUGUG
. . . UAUCACGG
. . . . . . . . UAC
. . C
. UGG
. . . CU
**i.
2
4
6
8
X
.:!';..' :..."
5
-
IM
Z-
N
X
o 1d
C .-endo
3
10 12 14 16 18 20 22 24 26 28 30
Residue
317K
1
0.9
0.8
0.7
N 0.6
Cl
C2 .-endo
0.5
'N
X ,-q
0.4
5
0.3
C 34-endo
C.-endo
n.2
ar
v
2
Figu
4
6
8 10 12 14 16 18 20 22 24 26 28 30
Residue
q the30-ntRNA at 298K
C6/C8('Base';blak solidl) andCi'(ibse'; gysolid lines)
stants
aregizen fpssile(bak
For de kp and dtetw a4antresidues, 3J(H,H2) awplzrga
20: S2parantefor
(abhze)and317K (4oz.
daisellui).
122
,
. .
Chapter 3: RNA Tetraloop Dynamics
Thus, the mean S2 of the stem residues, which do not exhibit increased dynamics, drops from
0.85+0.07 to 0.77+0.06 for the base and from 0.88+0.09 to 0.81+0.03 for the ribose moieties. In
agreement with these observation, the 3 J(H1.,H 2.) coupling constants for the two residues in the C 2 oz/o conformation, A15 and C16, remain the same at the two temperatures (Figure 20).
Of the residues in the uCACGg loop, positions L1 and L3 exhibit completely stem-like behavior at
both temperatures; L2 exhibits high flexibility both in its ribose and its base and L4 shows extreme
rigidityin both the ribose and the base at lower temperature. At higher temperature, the base but not
the ribose of this residue adopts increased flexibility to an extent of the average stem-like behavior.
In conclusion, the overall loop dynamics exhibit only minor changes at 317K compared to 298K,
signifying that the uCACGg loop structure is still intact at higher temperature.
Although the loop structure remains unperturbed at 317K, the regions adjacent to it are subjected
to dramatic dynamic changes. Thus, the base of G18, which constitutes one part of the closing base
pair, and the base as well as the ribose of the adjacent A12 on the opposite strand exhibit increased
flexibility.This demonstrates that while the actual loop structure of the uCACGg motif is still intact
at 317K, the stem adjacent to it has started to melt.
3.3.3.5 Motional implications
In combination with the imino proton line width data and the 3 J(H1 .,H2.) coupling constants, the
13 C model-free
parameters can be used to construct a dynamic model of the two tetraloops studied in
here. Translation of model-free parameters into defined motional models, however, has to be carried
out under careful steric considerations concerning the possible modes of motion in a given system.
Independent motions of the C-H 6 or C-H
vectors in the internally rigid base moieties have to be
due to rotations around the glycosidictorsion angle X.
In this case, the GAF-model can be applied, which translates S2 into a rotation around the Cq.N
axis (see Figure 9).
Figure 21 shows the extent of motion involved in a rotation around the glycosidic bond by +15 in
a canonical stem arrangement.
In addition to the motionally independent bases, all those involved in base pairs have been
modeled using the GAF-model. Since the flexibility of these moieties is thus considered to arise
exclusively from rotations around the glycosidic linkage the overall extent of flexibility might be
overestimated.
123
Chapter 3: RNA Tetraloop Dynamics
Figue 21:Fca
C
B
A
an.i'.
AC
-fomRNA
° .m
Bnym?';B& ' 15 dedat
In contrast to independent base dynamics, the motional basis of independent ribose as well as
simultaneous ribose/base flexibility is more difficult to assess. As a first approximation to the actual
motions, a simple 'Wobble-in-a-cone' model originating in the phosphodiester backbone has been
assumed in these cases. Due to the proximity of the base and the C1 ,H1 vector, independent ribose
flexibility cannot be due to internal ribose dynamics (Figure 22). Upon ribose repuckering from a C2 to a C3.-eo conformation, the internal ribose rearrangement causes C2.-H2, and CH
while the CH-vector
3
motions,
is not affected.
-4-
10
C2 .-endo
C3 .-endo
Figure 22:Rikbe pu&aigfian C2'-to C3 erk mtinm doesnotdangethe daew oto
and thenudecze.
f the C
-Hi waor
If both moieties are flexible, it has to be considered that the underlying motions can either be
concerted or independent or partly concerted (Figure23A-CQ.
This uncertainty arises from the varying sensitivity of the GH vectors in the base and the ribose to
different dynamic processes, depending on the nature, e. g. the direction, of the underlying motion.
124
Chapter 3: RNA Tetraloop Dynamics
A
B
C
2s
S2=S2
=S2
2 2 2
2
I
Figtr 23: G
V1V1/
1
S1
a (4, m uak (B)an pany
9
W/4
oN6
2*
21
2
S2 =S2lSl,
2
S4
I -'
I
td nmrfor thease atSI > s2 (0 of tomra
pawde 1 and2.
Since the extent of motional concertedness cannot be assessed from the data, the two dynamic
extremes, completely concerted motion (Figure 23A) and independent motion (Figure 23B) have
been determined in here. In cases, in which both moieties are flexible and base mobility exceeds the
mobility of the ribose (S2ibose>S2e, Figure 23Q, excess base dynamics have been fit with the GAFmodel. An additional simplification has been introduced for moieties exhibiting two fast time-scale
motions, for which accordinglytwo order parameters (S 2 and Sj ) are available.In these cases, both
the spatial restriction for the overall order parameter and the two order parameters separately has
been given. However, if the moiety in question was a ribose connected to a flexible base, only the
overall motional restriction (obtained from S2)has been propagated into the base in order to simplify
the analysis.
The results of the motional analysis of the 13C relaxation data are summarized in Table 13 and
Table 13 for the acUUCGgu loop and in Table 14 and Table 15 for the auCACGgu loop at the two
temperatures. In addition, the approximate base pair strengths extracted from the imino proton linewidth are given. Finally, the ribose pucker rigidity as obtained from 3 J(H1 .,H2 ) coupling constants is
also listed in Table 12 to Table 15. Base pair strength as well as ribose puckering are divided
semiquantitatively into different categories ranging from (+++) to (-) following the criteria described
in detail in the caption of Table 12.
125
Chapter 3: RNA Tetraloop Dynamics
298K
S3
S1
L1
L2
S2
Model
0
Base
a
pair
S2
Model
Ribose
ox ..
S2
S4
0.88
0.88
0.87
0.87
0.67
0.84
0.83
0.89
0.89
GAF
GAF
WGAF
GAF
GAF
GA
GAF
GAF
-
-
- 24 25o0~~
140
Rex
Base
L4
GAF
-
o,
L3
indep.
conc.
.
.
+++-
-
220
.
.
150
.
0.83
-
0.84
-
0.77
W
0.73
W
-
-
°
-
150
++
- -
0.82
-
100
.
+ +4
-
240
230 ...
°
-
120
70
-
-
++
+++
+++
0.82
0.80
-
0.81
-
Re,
Pucker
+++
+++
++
++
+++
+++
+++
Table 12 Dynanic infomution on the acUUCGgu loopat 298K The basepair strengthhas en assessed
firm
thepeak heightof the iino proton resonances.The basepair smrgts are categorized
asfdlaos: (+++) indicatesan
imino resonanceiamitie equal to the one of stable stem ridue; (++) is apliedfor only slightly deased
resonances
is labied am a residueis labed (+) f the rsonanceis st ngydsed
but sible,(-) is appliedin the absencef a
imino resonane The rigidity f the ribosepuker has been obtainedfrom the size f the 3J(H ,H2)
apling
1
wanstants.(+++)
is appliedforJ-crtplingslargerthan 10Hz and smller than 1Hz; (++) indiates a J-catplingof 8
to 10Hz or 1 to 3Hz; midues,for hich not tplingcotant
acol aqui are indicatedbya (-).
317K
----- -- - -)4
S3
S2
S1i
0.82
0.82
0.80
0.82
Model
GAF
GAF
GM
-
-
-
230°
,
00
Base
x160
Bae
Rex
Base
ase
pair
S2
Ribose
Model
0
a,,
Re
L2
0.77
0.75
0.81
0.83
GM
GAF
GAF
GAF
300
20 °
19 °
170
130
-
-
190
lops
2.2s
+
0.83
0.84
0.85
-
~
S4
GAF
-
++
S2
0.75
'
+++
L4
GM,W
130
180
L3
indep.
0.58
°
-
.
-
1.5s''
'
.+
++
+++
_______
-
0.77
0.75
0.81
0.81
0.82
W
230
W
250
-
-
-
-
-
-
-
-
-
+++
2.74s'
-
-
..
-
-
Rex .
Pucker
L1
-conc.
-
..
+++
.
.
+++
+++
+++
Table 13: Dymnic ifrontion an the acUUCGgu loo at 317K The basepair strengthbas beenassesedfrom the peak
height f the inino proton resonanes; The rigidty f the riose pudeker hs een btainedl from the seize f the 3J(H1,H2)
acling cwtants.
126
Chapter 3: RNA Tetraloop Dynamics
298K
S2
2
S
S2
Model
Base
----- ---- --+
------ -- --
S3
0o
Orx
S1
0.87
0.84
Pair
S
z
0.83
GAF
GAF
GAF
-
110
130
l
Base
L1
L3
L4
S2
S4
0.51
0.81
0.93
0.87
0.83
0.63
-
-
0.82
-
-
-
GAFW
GAF
GAF
GAF
GAF
GAF
350
-
190
15°
130
120
-
-
-
__
0.93
(250/160)
607ps
-.
0.92
S2
s2
Model
Ribose
00
ax
-
-
-
-
-
-
+++
Table 14: Dymnic ionntimn
height f the imino pron
0.96
0.86
0.89
0.67
-
-
-
-
0.82
-
-
-
972ps
+++
+ +
0.87
2 350 0
.......
++
0.55
W
4,
Pucker
idep..
310
90
x
q---++
0.9
170
L2
conc.
0.93
++
-
.
-
-
-
-
-
]
++
+++
on the auCA CGgu loopat 298K The basepair strnth hzs beenassessedfitnmthe peak
oresn s; The rigdity f the rike
pudeer has
auplingaotants
127
n ochtaimrd
firvmn the seize of thedx
3J(H1,H2)
Chapter 3: RNA Tetraloop Dynamics
S3
317K 317K
_
conc.
indep.
0.60
0.46
SI
0.80
2Sf~~~
GAF
GAF
GA
250
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-
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o;
~,
26 °
R,~sex
."c,,~_:_
34 °
______
140
I
:
:07
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:'0,
'0
0.58_ ~
S4
0.53
0.80
0.71
0.81
. - 0.73
GA
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-
-
350 '
'o
-
S2
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GAP'
200
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3180
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GA
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14
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0.77
(27/6,).-.
750ps
'.i:.
l7ps
...:'-:{.:..[:.:
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. 14....~mOr
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Base
Pair
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01:76-
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L2
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:'L'..-.'
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-
'." .
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Pucker
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-
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:
Table 15: Dymni ifanm
onon the auCA CGgu ko at 317K The basepir shth
hz beenassessedfrmnthe peak
heightof the im prton rarr;
The igidity of the ribse pucker hs I-an adiedfin
the seize of e 3J(H, H2)
cokql1zngcmtanl.
From this combined information, the detailed motional pictures of the two tetraloop motifs
presented in Figure 24 have been constructed. In here, concerted motions have been assumed.
The dynamics at 298K, at which both tetraloops are stable, can be viewed as the ground state
dynamics. Since the cUUCGg and the uCACGg motif exhibit almost identical structural features, it is
not surprising that their general ground state dynamic characteristics are similar. At a closer look,
however, the extensive analysis conducted in here allows for the identification of quantitative
differences in loop dynamics.
The most pronounced difference can be found in residue L2. While in the uCAGGg loop the whole
nucleotide exhibits fast internal motions, the flexibility of residue L2 in the cUUCGg loop is smaller
in its base and even smaller in its ribose moiety. In addition, the other residues show slightly different
dynamics in the two loops. Residue L3 is more mobile in the ribose moiety than in the base in the
cUUCGGgmotif, while it exhibits a rigid behavior in the uCACGg motif.
128
,.
.
Chapter 3: RNA Tetraloop Dynamics
cUUCGg
298K
5'
5'
317K
3'
uCACGg
,, -
L2
S1
S3
5
298K
3'
5'
Figur 24: Dynis
317K
3'
f the cUUCGg(top)and the uCA CGg (boawo loop at 298K (left) arrnd317K (night). The oazerall
dynaznis based on S2 are gizn For nvt,
zhrichpossessa 2 term d errtom 1, -r is gizen 7he 'Wobbir-a-re'
nmel
s depitad as mm, te GAF-rm ei as duradararnro.
129
Chapter 3: RNA Tetraloop Dynamics
Residue L4 is remarkable in that it represents one of the most stable residues overall in the
uCACGg loop at room temperature but has a slightly flexible base in the cUUCGg loop with a
ribose moiety of average stability. Imino proton line widths are in agreement with the ground state
dynamics in that the L1:L4 loop base pair and the two adjacent stem base pairs are intact. In case of
the cUUCGg loop, the increased mobility of riboses L2 and L3 in absence of ribose repuckering
points to increased flexibility of the entire phosphodiester backbone in the upper loop region.
At 317K, close to the melting temperature of the uCACGg motif, the relative dynamics of the
actual loop residues do not change for both motifs except for the slightly increased flexibility of the
base at position L4 in the uCAGGg loop. From this, it appears that all of the secondary structure
features of the cUUCGg and most of the uCACGg loop can still be present at 317K. Thus the
backbone interaction of the L3 base and the extensive base stacking of residues S1, L1 and L3
remain unperturbed (Figure 24).
While the dynamics of the loops are thus more or less temperature independent, the two base pairs
adjacent to it exhibit increased dynamics in the uCACGg loop, while the stem of the cUUCGg motif
remains unperturbed. At higher temperature, the two purine bases of the base pairs adjacent to the
uCACGg loop become about as flexible as the non-interacting residue at L2, while neither of their
respective base pairing partners show increased flexibility. Although one of the two base pairing
partners exhibits increased dynamics, in both cases, the hydrogen bonding arrangement remains at
least partially intact as apparent from the imino proton line width analysis. In contrast, although L4
exhibits only a minor increase in flexibility and the flexibility of L1 does also not change significantly,
the imino resonance of L4 has vanished completely at 317K. Thus, while the strong increase in
flexibility at positions S2 and S3 does not result in a complete disruption of the base pair
arrangement, the hydrogen bonds of L1i:L4 is broken without a significant increase in local dynamics.
The imino resonance of L4, however, reports only on the two base-to-base hydrogen bonds in the
L1:L4 base pair. The third hydrogen bond between the L4(0 6 ) and the ribose 02' of L1 can not be
monitored and could thus still be present. Furthermore, additional means of stabilization like base
stacking could keep the uCACGg loop structure intact.
130
Chapter 3: RNA Tetraloop Dynamics
3.4 DISCUSSION
In this work it has been attempted for the first time to characterize RNA dynamics in a way that
every residue is considered both in its ribose and in its base moiety. The approach was to measure
NMR autocorrelated relaxation parameters of C1, in the ribose and of C6 or C8 in the base moiety,
translate them into motional parameters by a common model-free analysisand interpret them further
by application of appropriate motional models. The aims were to
(i)
investigate the validity of the model-free approach for 13Crelaxation data in RNA.
(i)
scrutinize the relevance of the motional parameters obtained from the model-free analysis
in order to understand the determinants of stability as well as flexibility in a biologically
relevant system.
The dynamic investigation has been focused on the abundant RNA tetraloop family of the
sequence YNMG. Two members of this family, the exceptionally stable cUUCGg motif and the
uCACGg motif, which exhibits a similar fold but decreased stability, have been studied. In order to
determine the origin for the different loop stabilities on a molecular level, dynamic investigation was
carried out both at room temperature and around the melting temperature of the uCACGg motif.
The results of the dynamic analysis are discussed in the following both in regard to general technical
aspects as well as to the specific biological implications.
3.4.2 Model-free analysis
Application of the model-free formalism to a nucleus requires on the one hand isolated I-S spin
system type behavior and on the other hand knowledgeabout its CSA.
While the model-free formalism has been developed for isolated I-S spin systems, it has been
applied herein to study the dynamics of nuclei (C,, C6 and CQ)with not only a directly bound proton
but also an additional adjacent carbon spin (C2 , and C5 ). In agreement with earlier studies on small
DNA molecules [38, 39], it has been shown in here that the additional dipolar relaxation contribution
can be neglected for the size of the investigated molecules (Figure 14). Even for the larger of the two
molecules,the 30-nt RNA, the relative CCdipolar contribution amounts only to about 1%.
131
Chapter 3: RNA Tetraloop Dynamics
It could be shown here that in contrast to the neglectable CCdipolar contribution the CSA
constitutes a more crucial factor in the interpretation of
13C relaxation
rates (Figure 13). Especially
for the aromatic C6 and C8 , which possess large CSA values, knowledge about the size of the CSA is
of essential importance in order to interpret relaxation parameters. There are two studies on carbon
CSA values in nucleic acids available to date [25, 26]. From the analysis of the relaxation data it
resulted that it was in agreement with similar CSA values for C6 in both pyrimidines and for C in
both purines as reported in ref. [25]. Consequently, the
13C relaxation
study constitutes a measure to
assess the validity of these more or less theoretical tensor values for their application under
physiological conditions.
In addition to the absolute value of the CSA, also the orientation of the CS-tensor is relevant in the
case of anisotropic molecular diffusion. As demonstrated in here, for RNA, an axially symmetric
diffusion tensor has to be applied already for molecules as small as the 14-nt RNA in order to grant
accurate dynamic interpretation of the dynamic data (Figure 16). While in the model-free analysis an
axially symmetric CS-tensor with the unique main component oriented along the I-S internuclear
vector is assumed, all 13 C tensors in nucleotides have been reported to be fully anisotropic with their
principal axis tilted away from the CGHvector.
From the present analysis, the incorporated CSA values as well as the assumption of an I-S spin
system type behavior have been validated by the similarity of both the axially symmetric diffusion
tensor and the overall correlation time ;c obtained from the model-free analysis and the
hydrodynamic calculations as well as the ability to fit most of the relaxation data satisfyingly. In
addition, for both investigated molecules the stably base paired stem residues exhibit very uniform
dynamics as expected. Furthermore, at temperatures, at which both loop motifs are stable, their
dynamic profile is in agreement with their structural features. In spite of these in general reassuring
results, systematic deviations for the cytosine base dynamics have been noted, which are most likely
due to deviations of the actual CS-tensor from the applied one.
Model-free analyses of RNA will become more precise, if at the one hand CS-tensors of RNA
carbon spins are determined in more physiological systems such as directly on oligonucleotides in
solution. In principle, the CS-tensor can be determined from the geometric information conveyed in
multiple cross-correlated relaxation rates correlating the CS-tensor to the different neighboring
dipole-dipole vectors or from the anisotropic chemical shift information extracted from samples in
different alignment media. For proteins, a number of studies have been carried out along these lines
132
I......__
Chapter 3: RNA Tetraloop Dynamics
aiming at the determination of the amide
15 N
CS-tensor in solution [60-62], which could be
transferred to carbon spins in RNA.
For both the imino nitrogens and various carbon spins, S2 parameters have been determined in
earlier reports. From the 13Crelaxation rate analysis of the 14-nt RNA presented in here, averaged
S2
stem
values of 0.88 +0.003 and 0.81 +0.006 have been obtained for the base moieties at 298K and
317K, respectively. For the ribose, stem S2 parameters of 0.83_+0.001 and 0.87+0.001 have been
determined at 298K and 317K, respectively.
A
1 5N
relaxation analysis has been conducted on the 14-nt RNA studied here [59], reporting S2
values for the base paired guanosine and uridine residues (G1, G2, U6, G9, G10, Ul 1 and G12). The
S2 values obtained in the
in the
3C
1 5N relaxation
data analysis are compared to the ones acquired for the bases
analysis in Figure 25. Apart from G9, the order parameters in the
1 5N
study are
significantly lower (0.74 to 0.81) than the ones determined in here. Thus, the average S2 value of the
stem residues amounts to only 0.78 +0.01. While G9 seems to be the most stable residue according to
the previous report, it appears even slightly less stable than the stem residues in the "3C relaxation
data analysis. In addition, the relative stability of G10, U1l and G12 is reversed.
1
-
0.8
N
cn
stem
0.6
nfAvt/i Wl
loop
G G C A
I
I
1 2
CU
>
stem
U C G G U G
I
I
I
I
I
I
3
4
5
6
7
8
I
I
I
I
9 1011121
C C
I
I
314
Residue
Figwre25:S2uluesfinth 'C dandataaalysz
(Haae)at 298Kandfr '1N axaiodata(rey)potid in
ref [59.
133
Chapter 3: RNA Tetraloop Dynamics
It has to be noted that the
15N
analysis was carried out at 273K, while the
3C
data have been
obtained at 298K. Considering the differences in temperature, it is even more surprising that the l5 N
analysis reports higher flexibility. As for
13C
also ' 5N relaxation data analysis relies on knowledge
about the CSA. Thus, the respective CSA-values could be underestimated in our or overestimated in
the
15 N relaxation report.
Apart from that, it is possible that due to the different orientation of the
N-H as compared to the GH vector in the bases, "5 N and "3Crelaxation report on different motions.
In contrast, other studies on
3C relaxation
in different RNA molecules are more in agreement with
the data presented in here. Thus, average stem S2 values of 0.8 for C2 and C8 in purine bases have
been reported at 298K for the 16-nt iron responsive element RNA hairpin [37] and 0.7 to 0.9 for C6
and C in the 29-nt ATAR RNA [63], while slightly higher stem S2 values of 0.8 to 1.0 have been
determined for the adenine C2 in the 30-nt TAR RNA at 298K [42]. In small double stranded DNA,
averaged S2 values of 0.8 and 0.6 have been reported at 303K for the base and the ribose carbons,
respectively [35] indicating an increased ribose flexibility. In contrast, an averaged order parameter of
0.8+0.09 has been reported for thymine C1 . [39] and similar order parameters (0.8+0.1) were
obtained for both base and ribose carbons at 303K [32] as well as for two DNA octamers (-0.8) at
298K [34, 64].
3.4.3 Dynamics of the YNMG tetraloop motif
The starting point of this research was to understand the factors, which trigger RNA stability in as
much detail as possible. Dynamic investigations by NMR relaxation data analysis seemed appropriate
for this task, since they report on both the structure and the flexibility on a moiety as small as a bond
vector. In a simplified model, this vector can be interpreted as a dynamic reporter for a moiety like
the base or the ribose in a nucleotide. It has, however, to be pointed out that this simplification is
only valid, as long as the moieties are not subjected to internal motion. While this is always true for
the internally rigid base moiety, the ribose might undergo repuckering motions. In order to assert
ribose pucker rigidity,
3 C relaxation
data have been combined with J(H1 ,H2 .) coupling constants in
here. However, even in a rigid arrangement, the relaxation data is not obtained for the whole moiety
but for a given CH vector, which in turn is sensitive to a different degree to different motional
events depending on their relative orientation. Therefore, the amount of flexibility extracted from
relaxation data does not necessarily account for all the dynamics of a moiety. In order to improve the
134
Chapter 3: RNA Tetraloop Dynamics
dynamic investigation, multiple dynamic probes should be used for a given moiety. This, however,
implies lengthier data acquisition and analysis procedures and might even render the analysis more
confusing, since different vectors report on different motions without provision of information on
the underlying motional events.
3.4.3.1 Ground state dynamics
Comparison of the cUUCGg and the uCACGg motif at room temperature reveal an overall similar
but not identical dynamic picture. Despite its decreased thermostability, the uCACGg motif is not
the more flexible one. Nevertheless, it contains the most mobile residue at position L2, while the
remainder of the loop is rigid. In contrast, the cUUCGg motif exhibits smaller,yet more delocalized
dynamics in the upper two residues (L2 and L3). These differences in the dynamics could arise from
slight variations in bond lengths or in the extent of base stacking interactions due to the difference in
the primary sequence.
One of the questions in the study of dynamics is to which extent they are actually coupled to
structure. In case of the actual loop region of both motifs studied in here, the dynamics are in
complete agreement with the structure, pointing to a close structure-dynamics relation at room
temperature. To what extent the differences observed between the two motifs can be attributed to
deviation from the strict structure-dynamics relation or arise from slight structural differences is
debatable. The effect on stability of single or multiple nucleotide replacements in the UUCG motif
has been extensively studied [12]. In the present case, the exchange of a uridine for a cytosine at
position L1 signifies the exchange of an oxygen for an amino group, both of which are not engaged
in stabilizing interactions. In addition, the exchange of a uridine for an adenine at position L2 does
not involve any change in secondary structure interactions. Thermodynamic studies have shown,
however, that both nucleotide exchanges are unfavorable in regard to the thermostability, both
separately and in connection with each other. The gain in stability is, however, only marginal. Thus,
the melting temperature of the cUUCGg loop is 72.9°C, while the one of the uCACGg motif
amounts to 69.6°G If L2 of the cUUCGg is replaced by an adenine (cUACGO), the melting
temperature drops by 2°0 G Under the assumption that flexibility can be correlated with stability, the
increased dynamics of an adenine at position L2 as compared to an uridine offers a first insight into
the reasons for these differences in thermostability upon exchange of a base, which is free from
interactions. Thermodynamic studies on different tetraloops [12] also demonstrate that the effects of
nucleotide exchanges are not independent, but vary within different sequence context. Thus, while
135
Chapter 3: RNA Tetraloop Dynamics
an adenine at position L2 represents the least stable choice in a CNCG loop, for an UNOG loop, the
cytosine variant is the least stable one.
Alternatively, the differences in loop dynamics could be also attributed to remote effects of the
closing base pair. In contrast to an CG Watson-Crick base-pair, a U:G wobble base pair possesses
only two hydrogen bonds and causes local differences in the helix structure [1], which could
propagate into the loop and account for the observed dynamic differences.
One of the attractive features of dynamic investigations is their content in both dynamic as well as
structural information, which can serve to report not only on actual changes in stability but also on
dynamics in an otherwise stable arrangement. This can help to identify unstable structural features
within a structure even under stable conditions. From the comparative study at a temperature, at
which both loops are stable, no putative later melting sites in the uCACGg motif can be identified,
since the dynamics are completely congruent with the structural features.
3.4.3.2 Melting of the uCACGg motif
While the dynamics of the cUUCGg motif remain more or less unaltered at higher temperature,
the uCACGg motif exhibits large dynamic changes in several residues. Apart from a general gain in
flexibility, the dynamic profile of the loop remains almost unperturbed at higher temperature and
most of the initial stabilizing interactions are still possible. Hence, as expected, the sequence
difference in the actual loop region does not constitute the deviation in stability from the cUUCGg
motif. In contrast to the actual loop, one of the residues of the closing base pair and one adjacent to
it acquire flexibility equal to the dynamics of the free base of position L2. The stability of the
respective base pairing partners and the presence of, however, weakened hydrogen bond in the base
pair point to a more or less unperturbed structure. This signifies, that at 317K the initiation of
uCACGg melting is observed. From the combined data presented in here, the structural and dynamic
features of the uCACGg motif at the initiation of melting given in Figure 26 have been extracted.
In contrast to the situation at room temperature, at 317K, the observed dynamics deviate from the
structure. While hydrogen bonds can still be observed for both the closing base pair S1:S2 and the
adjacent one S3:S4, the flexibility of one base pairing partner in each base pair is highly increased. In
contrast, L1 remains completely rigid and L4 gains only slightly in flexibility although no hydrogen
can be observed for this base pair. Only from the dynamic information it becomes evident that the
two base pairs adjacent to the loop and not L1:L4 constitute the primary melting site.
136
..
.
Chapter 3: RNA Tetraloop Dynamics
7;VI
ulual y
-
aJ
ma
melting
site
Primary
melting
site
*
%
5'
3'
FiguWe26: lMdg de EuCACGglp. A nives
andtm sales qfrtr
amre
gn
rie nety oS2 (shd dakgry).
No data wdd be aimd fr the
Due to signal overlap, no information on the ribose moiety of S2 could be obtained. This increases
the difficulties in elucidating, which of the two base pairs (S1:S2 or S3:S4) constitutes the origin of
melting. Although the overall S2 values for the two flexible base moieties of resides S2 and S3 are
very similar (0.53+0.06 and 0.46+0.01), they exhibit dynamics on widely different time scales.
Whereas S2 shows motions on two fast time-scales (< 10lps and 1800ps), the flexibility of S3 is due
to only one high amplitude, fast time-scale (< l10ps) motion. Apart from that, the imino proton line
width indicates about the same strength in residual hydrogen bonding. Hence, although differences
in dynamics can be detected, it is not possible to narrow the identity of the melting initiation site
further down to any one of the two base-pairs adjacent to the loop, since no information on the
motional bases of the observed dynamics can be obtained from the data. The slightly increased
dynamics of the L4 base and the absence of the L1:L4 base:base hydrogen bonds indicate that L4
constitutes the secondary melting site, through which the stem melting is propagated into the loop.
137
Chapter 3: RNA Tetraloop Dynamics
In conclusion, the dynamic investigation of the YNMG
tetraloop motif presented here
demonstrates that loop stability is not only triggered by the nature of the closing base pair but also
depends on the one preceding it. While the significance of the closing base-pair as a determinant for
loop stability has been long recognized and extensively studied [12, 19, 65], the effect of more
remote base pairs has not been acknowledged so far.
3.4.3.3
Biological implications
Why does the same RNA secondary structure motif appear in so widely different sequence context
with such a difference in stability? Why, if a low melting temperature is required for the SLD in the
Coxsackievirus, is the thermostable CAGG tetraloop not replaced by a less stable loop motif?
An explanation for the necessity of an unstable member of the YNMG motif at this position on
the viral genome could arise from the different requirements in this sequence. On the one hand, the
recognition of SLD bythe viral precursor protein 3CD P° is purely structure based [1]. Thus, protease
binding affinity remains unaffected, when the tetraloop is replaced by a UGCG motif [1]. On the
other hand, the secondary structure of SLD has to melt in order for the replication of the viral
genome to take place. It follows that while SLD has to be highly structured for protein-recognition,
the increased stability accompanying the structural content is not favorable for the low stability
required for replication (Figure 27). In addition, none of the loop residues is especially conserved
[20], the most conserved being the Gsy at position L4 (85%), followed by position L3 (73%
). The
A at position 2 occurs only in 52%, while the most frequent nucleotide at position L1 is, U, is
conserved only in 48% of the sequences (C is found in 27%). In contrast, the G of the closing (97%)
base pair and the two adjacent base pairs (90% and 100%) are highly conserved.
The conserved stem-residues contain also the residues, which acquire increased mobility at higher
temperature, implying that the decreased stability of the closing- and the adjacent A:U base pairs is
biologically relevant. It is possible that the stem melting is relevant for initiation in viral RNA
translation.
Apart from the recognition by the 3CDP ° protein, the YNMG motif at this position might be
required for reliable folding of the highly structured 5'-non-translated region of the viral genome.
138
. .
Chapter 3: RNA Tetraloop Dynamics
A
B
5'
Figure 27: Tfd
3'
-
5'
3'
dernnd onSLD. Suaal stabilityisnrtdnfor bin or3C
(4); sm
for stsraraxpdwi
g eplicaionofdx Hralram (B).
ralcfiexibiltys m d
3.5 OUTLOOK
This study has for the first time presented an almost complete dynamic picture of an abundant
RNA secondary structure motif. Comparativestudies of the cUUCGg and the uCACGg motif of the
YNMG tetraloop family demonstrate that loop stability is triggered not only by the nature of the
closing base-pair but also by the identity of more remote base-pairs at least one position away from
the loop. This stability fine-tuning seems to be used by nature to decouple structural and stability
requirements in order to adapt hairpin structures for opposing purposes.
The study presented constitutes the first step in investigating the effects of remote base pairs on
loop thermodynamics. In the future, systematic studies to this purpose should follow.
139
Chapter 3: RNA Tetraloop Dynamics
3.6 REFERENCES
1.
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ue (Cam), 12, 237-248.
Cloverleaf RNA and Its Interaction with the Proteinase 3G S
2.
Noller, H. F. (1984). Structure of ribosomal RNA. A
3.
Tuerk, C; Gauss, P.; Thermes, C; Groebe, D. R.; Gayle, M.; Guild, N.; Stormo, G.;
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4.
Uhlenbeck, O. G (1990). Tetraloops and RNA folding. Nate,
5.
Jaeger, L.; Michel, F.; Westhof, E. (1994). Involvement of a GNRA tetraloop in long-range
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6.
Murphy, F. L. and Cech, T. R. (1994). GAAA tetraloop and conserved bulge stabilize tertiary
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7.
Liao, X. B.; Brennwald, P.; Wise, J. A. (1989). Genetic analysis of Schizosaccharomyces
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8.
Selinger, D.; Liao, X.; Wise, J. A. (1993). Functional interchangeability of the structurally
similar tetranucleotide loops GAAA and UUCG in fission yeast signal recognition particle
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9.
Zwieb, C (1994). Site-directed mutagenesis of signal-recognition particle RNA. Identification
of the nucleotides in helix 8 required for interaction with protein SRP19. Eurj Biem 222,
885-890.
10.
Woese, C R; Winker, S.; Gutell, R. R. (1990). Architecture of ribosomal RNA constraints
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11.
Varani, G. (1995). Exceptionally stable nucleic acid hairpins. Annu Rev Biqb
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Proctor, D. J.; Schaak, J. E.; Bevilacqua, J. M.; Falzone, C J.; Bevilacqua, P. C (2002).
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13.
Varani, G.; Cheong, C; Tmoco, I., Jr. (1991). Structure of an unusually stable RNA hairpin.
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14.
Allain, F. H and Varani, G. (1995). Structure of the P1 helix from group I self-splicing
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Ennifar, E.; Nulin,
Ehresmann, B.; Ehresmann, C; Nikonov, S.; Dumas, P. (2000). The crystal structure of
UUCG tetraloop. JMdolBid, 304, 35-42.
16.
Nissen, P.; Hansen, J.; Ban, N.; Moore, P. B.; Steitz, T. A (2000). The structural basis of
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140
Chapter 3: RNA Tetraloop Dynamics
17.
Wimberly, B. T.; Brodersen, D. E.; Clemons, W. M., Jr.; Morgan-Warren, R. J.; Crter, A. P.;
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Paul, A. (2002) Possible unifying mechanism of picornavirus genome replication, in Mdar
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Schmidt, P. G.; Sierzputowska-Gracz, H; Agris, P. F. (1987). Internal motions in yeast
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Gaudin, F.; Paquet, F.; Chanteloup, L.; Beau, J. M.; Nguyen, T. T.; Lancelot, G. (1995).
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Olsen, J. I.; Schweizer, M. P.; Walkiw, I. J.; Hamill, W. D., Jr.; Horton, W. J.; Grant, D. M
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Williamson, J. R. and Boxer, S. G. (1989). Multinuclear NMR studies of DNA hairpins. 1.
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Biahernity, 28,2819-2831.
Kojima, C; Ono, A.; Kainosho, M.; James, T. L. (1998). DNA Duplex Dynamics: NMR
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Spielmann, H P. (1998). Dynamics in psoralen-damaged DNA by 1H-detected natural
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Lane, A. N. (1991). Solution conformation and dynamics of the octadeoxy-nucleotide
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Hall, K. B. and Tang, G (1998). 13 C relaxation and dynamics of the purine bases in the iron
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Zimmermann, G. ; Shields, T. P.; Jenison, R. D.; Wick, CG L.; Pardi, A. (1998). A
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Schweizer, M. P.; Olsen, J. I.; Stolk, J. A.; Lee, Y. C; Reeves, P. VM.;
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Chapter 3: RNA Tetraloop Dynamics
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144
Chapter 4: Structure of the TCR C-chain
CHAPTER 4
Structural Investigation of early events in T-cell
Activation.
NMR-investigation of the T-cell receptor 4-chain in solution
and bound to lipid micelles.
145
Chapter 4: Structure of the TCR C-chain
Specific Aims
The goal of this research was to contribute structural information from NMR experiments to a
functional investigation of the early events of T-cell activation upon antigen interaction. The
investigation was focused on the cytoplasmic portion of the T-cell receptor (TCR), its C-chain, the
putative mediator between the extracellular antigen and the intracellular signal transduction cascade
leading to T-cell activation. Earlier studies resulted in a T-cell activation mechanism that proposed a
TCR state dependent lipid incorporation propensity of the
-chain accompanied by a folding
transition [1]. In order to support this proposed mechanism, standard protein NMR assignment and
secondary structure elucidation techniques have been applied to TCR g bound to detergent micelles
in order to obtain the structural characteristics of this folding transition in a residue resolved manner.
In addition, the assignment of free TCR 4 constitutes the basis for residue specific investigations on
its interaction with the HI-V-proteinNef, the interaction of which with TCR ~ has been shown to
impair T-cell function through a number of different mechanisms [2-4]. In addition the recently
reported dimerization of TCR 4 [5] has been investigated.
TABLE OF CONTENTS
4.1
INTRODUCTION
4.1.2
4.1.2
4.1.3
4.1.4
4.2
4.2.2
4.2.3
4.2.4
4.2.5
4.2.6
4.2.7
4.3
.................................................................................................
148
148
T-Cell Activation ................................................................................................................
149
The TCR -chain ...............................................................................................................
TCR /Nef interaction....................................................................................................150
Protein backbone resonance assignment........................................................................152
MATERIALS AND METHODS ............................................................................ 154
154
Protein expression..............................................................................................................
154
Backbone assignment of free TCR ...............................................................................
Concentration dependent experiments ...........................................................................155
Temperature dependent experiments ..............................................................................155
155
~/Nef interaction studies ..................................................................................................
156
TCR in LMPG micelles..................................................................................................
RESULTS ................................................................................................................
157
157
4.3.2
TCR in solution...............................................................................................................
157
4.3.2.1
Backbone Assignment ...................................................................................................
162
4/Nef-interaction ...........................................................................................................
4.3.2.2
146
Chapter 4: Structure of the TCR ;-chain
4.3.2.3
/~-Dim erization ............................................................................................................
166
4.3.3
TCR 4 bound to LM PG-niicelles .....................................................................................
169
4.4
CONCLUSIONS .....................................................................................................
174
4.5
REFERENCES .......................................................................................................
176
147
Chapter4: Structure of the TCR C-chain
4.1 INTRODUCTION
4.1.2 T-Cell Activation
T-cell activation is initiated by binding of its surface located T-cell receptor to antigens presented
by major-histocompatibility complex (MIC) proteins on the surface of body-cells. This process
involves the interaction of several associated proteins on either cell surface (Figure 1).
nntiaen nresentinacell
TC
Tcell
.,"-
U
Figur 1:T-d mweptorantintml c.
Antigen binding is followed by TCR clustering [6, 7]. This leads to multiple phosphorylations of the
TCR C-chain cytoplasmic domain at specific so-called ITAM sequences gmmunoreceptor
Tyrosine-
based Activation Motifs) by the tyrosine-kinase Lck and/or other members of the tyrosine-kinase
family [8-10]. Phosphorylation of TCR ( constitutes the starting point of the intercellularsignaling
cascade resulting in T-cell activation. After phosphorylation TCR ~ binds to and activates the
cytoplasmic tyrosine kinase ZAP-70 (or Syk) through interaction with its tandem SH2 domains [11].
Bound ZAP-70 then activates other tyrosine and serine-threonine kinases, induces phosphatidyl
inositol turnover and binding of adapter molecules as well as other processes in the early phase of Tcell activation [12, 13].
148
Chapter 4: Structure of the TCR 4-chain
4.1.2 The TCR 4-chain
The invariant C-chain of the T-cell receptor is a 164 amino acid single-pass transmembrane protein,
which exists as a disulfide-linked homodimer within the receptor. Its Gterminal cytoplasmic domain
of 112 amino acids carries three ITAM motifs of the consensus sequence Yxx(L/I)x,
8
Yxx(L/I),
where x can be any amino acid (Figure 2). During T-cell activation, the ITAM motifs are
phosphorylated to a varying degree at different tyrosine residues.
;
ITAM1
GSIRVKFSRSADAPAYQQGQ
NQ. LI
'L//DKRRGRDPEMGGKPRRKNPQEG
10
20
30
ITAM2
tQKDKKQA
70
40
50
60
ITAM3
GMKGERRRGKGHDGL|STZlTKDHMQALPPR
80
90
100
110
Figure 2: Pirwy some ef x 7TCR -chinOtpinic damn asit hasbLenstudid
Ther7x
e s
canies m
an acds of
its tramnwraw dmin at its N-tiu.
1Thx
dshed wd1 ln nwks he gmg ce
tclasrnic
doami Thet ITAMseques amre
rimt ndark grey(YxxL-rr andligtg y(
eiablienkersoxts).
Tyrsit
msduhi, can
ateidng Tcd acti , arshan l&tgy
The mechanism, by which TCR clustering is linked to intracellular C-chain phosphorylation, is not
yet well understood. Unlike other receptors activated by clustering, TCR g does not possess any
intrinsic enzymatic activity or associated kinases, nor do the TCR antigen binding domains appear to
undergo any significant ligand-dependent conformational changes, which could account for signal
mediation across the membrane. Amongst the various mechanisms proposed for signal transfer are:
subtle conformational changes below the resolution of the available crystal structure [14], allosteric
changes in the TCR due to ligation [15], co-localization of co-receptor-bound
kinases with their
substrates on TCR cytoplasmic domains (as both bind to MHC proteins) [16] and large scale
reorganizations of membrane proteins [17].
Recently, it was found by the group of Prof. Stem that the intrinsicallyunstructured TCR 4-chain
undergoes a structural transition to a more a-helical state upon interaction with acidic lipids [1]. This
lipid induced folding transition as well as the immunity of TCR ~ to phosphorylation by intracellular
kinases in its lipid bound state, has led to the proposal of the signal transduction mechanism
presented in Figure 3. In this model, TCR ; is embedded in the membrane with its ITAM sequences
sequestered away from any cytoplasmic kinase in the resting T-cell. Upon binding of antigen loaded
149
Chapter 4: Structure of the TCR -chain
MHCQ,TCR clustering results in a steric release of the 4-chain from the membrane and thus renders
it amenable to phosphorylation.
This T-cell activation model has been the outset of a collaboration between our group and the one
of Prof. Stem aimed at the structure determination of the 4-chain cytoplasmic domain in its lipid
bound state by NMR. The objective of the project was to investigate whether the structure of
LMPG- bound ~ provides an explanation for the impossibilityto phosphorylate this species.
Resting
State
Receptor
Clustering
Activated
State
Immune Response
Figume3: Ped
Structure adoption of TCR
sigrp tranrsdmin rdmsm for Tell aition
mniatd by TCR 4
upon micelle-interaction was probed by a chemical shift index (CSI)
study of the HA chemical shift changes. As described in the introduction, the a CSI is sensitive to
ac-helical and -sheet deviations structure deviations from the random coil.
4.1.3 TCR /Nef interaction
Nef (negative factor) is an accessory protein found in human immunodeficiency virus (HIV) type 1
and type 2 as well as in simian immunodeficiency virus (SIV). Although it does not possess any
150
Chapter4: Structure of the TCR 4-chain
intrinsic enzymatic function and is not required for virus replication, it is mandatory for achieving
and maintaining high viral loads in ziw [18]. It does so by interacting with various cellular signaling
pathway as well as immune cell specific proteins. One of its effects is a reduction in the number of
CD4 receptors on the surface of infected cells by binding to the cytoplasmic domains of CD4 [19].
CD4 receptors play a role in preventing reinfection by budding virions. Stimulation of the viral
replication has been linked to the interaction of Nef with the SH3 domain of Src family tyrosine
kinases [20-23] as well as a serine/threonine
of Nef to TCR
C
kinase [24-26] and protein kinase C theta [27]. Binding
has various different effects: It has been connected to upregulation of the so called
Fas-Fas ligand interactions, which lead to apoptosis [4]. In addition, interaction between Nef and
4
results in the downmodulation of the complex formed by the TCR and CD3 and thereby reduces the
availability of CD3 for stimulation by cross linking with anti CD3s antibodies [2].
By means of multiple deletion mutants, two independent Nef binding sites have been identified
within the cytoplasmic domain of TCR ~, each of which overlaps with an ITAM sequence (Figure 4)
[3].
Transmembrane Domaine
N
o
Cytoplasmic Domaine
ft
-ITAM 1
ITAM 2
ITAM 3
\
3
24
34
_
74 //?79
*
Figure 4: TCR /Ne f Sveraawnsites as detemi by ddain and mdte alnm sutm
interaazn sies ardual
ae shon ekI
Residuesdt am dal for Nef burig
sed n
nragsi
aremhigbiMi The ricke,wibm of the ytqolasnicdonflow
the wmlae m
For this study, only the
FC
115
rrnagembis
[3]. The
aim s',
d in the
nerf the
120 amino acid core domain of the -27 kDa SIV Nef has been used,
since the full length protein tended to aggregate at higher concentrations. Simian Nef was used
instead of the HIV Nef because of better expression yields.A binding constant of -0.3 to 2 ILMof
SIV Nef to TCR ~ has been determined by surface plasmon resonance by our co-workers.
In order to characterize the binding site of Nef to TCR 4 by NMR, H and 15N chemical shift
perturbation studies upon addition of Nef to TCR ~ were carried out. This required the backbone
resonances assignment of the free C-chain, which as well as the one of the lipid bound form was
obtained from standard protein assignment experiments.
151
Chapter4: Structure of the TCR C-chain
4.1.4 Protein backbone resonance assignment
Protein resonances assignment starts with and is often limited to the assignment of the backbone
H, C, HIF, N and C resonances within each residue. The assignment strategy for a known amino
acid chain follows two basic principles, connectivity and resonance pattern specificity. Connectivity
between residues can be established by exploitation of interresidual through bond (scalar couplings)
or through space (nOe) interactions. In addition to resonance connectivity, a varying degree of
residue type information is conveyed in these experiments depending on the uniqueness of the
resonance pattern. Obviously the extent of amino acid type specific information decreases with the
distance from the side chain from dC and C to N and C. While residue specific information is vital
to pin assignments to the amino acid sequence, it causes resonance overlap for stretches of identical
or chemically similar residues. In order to achieve optimal assignment, purely sequential experiments
are complemented with those conveying sequential as well as residue specific information and
exclusively residue specific experiments. In this report, among the variety of available NMR
assignment experiments, 3D-HNCOCO or 3D-HNCACO spectra were recorded for purely
sequential assignment, 3D-HNCACB spectra complemented the sequential assignment as well as
provided residue type specific information and 3D-HCCC)NH
information.
spectra yielded residue type specific
The correlation pathways and the resulting spectra of these NMR assignment
experiments are depicted in Figure 5. The nomenclature of the experiments reflects the underlying
magnetization transfer. The underlined nuclei are the ones, which chemical shift is evolved within
the experiment.
152
Chapter 4: Structure of the TCR c-chain
HNCACO
R
R
I
I
I
IC 55
C
1 13
//-N- CC'
IN
H
11
8
R
~~~~~~~~~~~I
C'--C-
55C
C'- NIa
H
i-1
C'-//
CI
. ..
IN
...
1+1
i
:
i-1
: i-2
N
Hi-1
HNCOCO
R
R
R
I
I
I
C1
4
C
i+1
4C
R. A
& .............. ....
I
1I-N- Ca-C C'N-CaN- C'-N NC- C-1
15
H
HN
i-1
"lI
C
...
i. !
15
....
..i..
.
. i-2
i-1
i+1
i
_
.
N
Hi-l
HNCACB
R
R
R
I
I
I
35 C
//- N
35
Ca-
...---
HiN
c--
*C'-N
*Ca~ C -//
IN1
HN
H
i-1
i+1
i
HCCCONH
H
R
H Ce
HNH55
0
R
I
I
Cj
15
I//-N- C- C'-N!i-1
R
Ca- C'-
H
i-!
N-
C-
C'--//
iN
H
il-
i
N
Figure 5: Rernt
armus; bken
arrm
NMR hkdeb
sigrf
transfer
H1
assignmnt epennt.
N
lHi
Resonane transfer by scalar aplings is iicated by slid
thrgh strong mopling by TOCSY
rrxing squers.
The mopling camtants for the
transferavregewn in gy The spins detea in the pexnt
are underlinedin the e xn t narm and iicated by uhite
baes. The resonanepattens zeithinthe HN-C or HN-H aliphaticplanesare shozensdemzticaJlyfor
the adaent ridues i
and i-1for ehx exnnent Hal' in thebhottonpad sigrif thealiphaticside-dhain
hb)drgr.
153
Chapter 4: Structure of the TCR ~-chain
4.2 MATERIALS AND METHODS
4.2.2 Protein expression
The whole protein expression and purification procedure was carried out by A. Sigalov in the
laboratory of our collaborator Prof. Stem. 15N- as well as "5N,
3C
labeled cytoplasmic domain of
TCR g of the sequence shown in Figure 2 was expressed following the protocol given in ref. [5]. The
core domain (residues 92 to 236) of SIV Nef was also expressed in Prof. Stems laboratory.
4.2.3 Backbone assignment of free TCR C
The backbone assignment of the free TCR ~ in solution has been carried out using a fully 3C, 5 N
labeled 1mM sample in 20mM phosphate buffer at pH 6 and 278K and 310K. In order to improve
the resolution in the
5N
dimension, semi-constant time versions of standard 3D-HNCACB [28],
3D-HNGOCO and 3D-HCCCONH
[29] NMR experiments were carried out on 600 MHz Bruker
spectrometers equipped with a IH,{ 3 C/ 15N}-triple resonance probe or a H,{13 C/15 N}-triple
resonance cryoprobe. The experimental conditions are summarized in Table 1. In all experiments, C
and C coupling to nitrogen was removed by application of selective refocusing pulses (G3.256,
5001xs)centered at 38 ppm and 174 ppm in the nitrogen evolution period. Side chain protons were
decoupled from carbons by the same pulse centered at 38 ppmr. CC TOCSY transfer was achieved
by application of a WALTZ sequence of 97ms using a 90{s carbon pulse. The experiments have
been processed using Bruker xwinnmr software. The resonances assignment has been carried out in
the program xeasy. 'H chemical shifts were referenced directly to 3-trimethylsilyl propionic-2,2,3,3-d 4
acid sodium salt (MSP). 13C and ' 5N chemical shift referencing was deduced indirectly from the H
referencing.
F1
Nuc
TD
T
F3
F2
SW OFF Nuc TD
SW OFF Nuc TD
SW
T
OFF
[K]
310
278
16
16
HNCACB
1H
1024
13C
110
60
41
15N
64
21
118
1H
1024
9
9
4.7
HNCACB
HNCOCO
4.7
13C
15N 48
20
4.7
4.7
13C
174
15N
28
19.6
310
8
1H
11
11
117.2
117
HNCOCO
1024
1024
60
11
40
1H
62
90
5.5
174
1sN
64
21.1
118
278
8
HINCOCO)
'H
1024
11
4.7
13C
5.5
174
15N
64
21.1
118
310
8
HCCGONH
1H
1024
4.7
1H
100
100
128
8.5
4.7
15N
64
22.5
118
310
4
Table 1: Badeb
j8.5
13C
pnts
7Th 4. T he detect mdeus (Nut), the mirr
refnlec
e faiemr y (OFF) inppmfir & dinrm onef h epetinrnt ae gen
assigmrentexpermnr for fie
(iD) and the spearaluiah (SW) and dx
thogd"r
w dx nwir jtramww ae refr
z&
vvnm (NS).
154
Chapter4: Structure of the TCR ~-chain
4.2.4 Concentration dependent experiments
'H," 5 N-HSQC spectra with concentrations of 0.4mrnMand 0.01mM "5 N-labeled TCR
20mM
phosphate buffer at pH 6 were obtained in subsequence to each other on a Bruker 600MHiz
spectrometer equipped with a 1 {C/
5 N}
cryoprobe at 278K. For both experiments the proton
and the nitrogen offset was set to 4.69ppm and 118pprm,respectively. 62 complex points were
acquired in the nitrogen dimension with a spectral width of 21ppm. 16 transients were obtained for
each increment in the indirect dimension for 0.4 mM sample, while 786 transients were used for the
0.01mM sample.
4.2.5 Temperature dependent experiments
In order to transfer the assignment of free TCR ~ from 278K to 310K, 1 H,N-HSQC
spectra were
obtained out in steps of 10 C or 2°C depending on signal overlap. The spectra were recorded on the
13CQ
5N-labeled
1mM of TCR 4 sample used in the backbone assignment experiments on a Bruker
600 MHz spectrometer with a
3 C/ 5 N} probe.
LH,{
The
5 N-offset
was set to 117.5ppm, 256
complex points were acquired with a spectral width of 23ppm in the ' 5N-dimension. In order to
suppress resonances splittings due to carbon-nitrogen scalar couplings in the indirect dimension
within the doubly labeled sample, selective carbon pulses (G3.256, 250,us) were applied in the
nitrogen evolution period on C. (38ppmrn,
on resonance) and C (offset of 21000Hz from the carbon
carrier frequency). 4 transients for each increment in the indirect dimension were applied for spectra
between 278K and 283K and 8 for spectra between 285K and 310K
4.2.6 /Nef interaction studies
In order to study binding dependent chemical shift perturbations, H,1 5N-HSQC spectra were
recorded at 298K for free
5N
labeled 4 and 15N labeled g in the presence of a twofold excess of
unlabeled SIV Nef core domain. Measurements were carried out in 10mM phosphate buffer in the
presence 100mM NaCI at pH7. The concentration was 0.4mM for free
and 0.2mM
in the
presence of Nef. The experiments were carried out on a Bruker 600MH-izspectrometer equipped
with a
H,{ 13 C/l5 N} triple resonance probe. The nitrogen carrier frequency offset was set to
117.Sppm, the spectral width was set to 25ppm; 128 complex points were acquired with 8 and 32
155
Chapter 4: Structure of the TCR ~-chain
scans for each increment in the indirect dimension for TCR ( alone and in the presence of Nef,
respectively.
4.2.7 TCR C in LMPG micelles
1-myristoyl-2-hydoxy-snglycero-3-[phospho-rac-(1-glycerol)]
(LMPG, Figure 6) was purchased
from Avanti polar lipids.
OH
H.
,OH
0~~~~~
Figure 6: LMPG
The TCR ~:detergent ratio was optimized for sensitivity and resolution in 'H1 5 N-HSQC spectra
using 0.4mM '5N-labeled c-chain with an 150 to 600 fold excess of LMPG. With the optimized TCR
~:LMPG ratio, the temperature was optimized within a range of 293 to 310K using 'H, 5N-HSQC
spectra. Assignment experiments were carried out using a l3 C, 5 N-labeled sample of 0.4m
TCR ; in
20m phosphate buffer (pH 6.0) at 310K. 3D-HNCACB, a 3D-HNCAGO, a 3D-HCCCONH and
a 3D-HCCCONH
[29] were obtained with the parameters summarized in Table 2. In the
HCCCONH and the HCCCONH the
C TOCSY-transfer was achieved by application of a
DIPSI2 cycle of 11.5ms with a 25ps pulse.
F1
F2
F3
NS
OFF Nuc TD SW OFF NS
Nuc
TD
SW OFF Nuc TD
HNCACB
1H
1024
14
4.7
15N
48
23
117.5
13C
80
60
43
16
HNCACO
1H
1024
14
4.7
15N
48
23
117.5
13C
100
6
174
8
HCCCONH
H
4.7
4.7
36
48
23
21.5
117.5
117.5
13C
80
64
65
6
40
IH
14
14
15N
HCCCONH
1024
1024
16
16
Table 2: Backdebor
assigm
15N
expemns for CR
SW
1H
botm to LMPG. The the
cwlc pom (D) and iespearalueidh (S W and ie mer p
aregiun tagedr wth the mniber cf trarw& atardieftr
airfor hinm
156
(FF)
(NS).
inppnfr
3
deae
mides (Nu, the mw-er f
ai
izon cf the
cXm
Chapter 4: Structure of the TCR C-chain
4.3 RESULTS
4.3.2 TCR g in solution
4.3.2.1 Backbone Assignment
The optimal temperature for NMR backbone assignment experiments on free TCR ~ was assessed
1 5 N-HSQC spectra.
from the general appearance as well as the sensitivity of H,
Inspection of a
temperature range between 278K and 310K resulted in a continuous worsening of the sensitivity
with increasing temperatures. Figure 7 shows
Hl,N-correlation spectra (HSQQ) at 278K and 310K,
demonstrating overall decreased resolution and sensitivity due to resonance broadening at higher
temperature. In addition, some resonances have broadened to a point where they are hardly
detectable at all. Taking into consideration that the spectrum at 310K has been obtained with double
the number of scans, the signal to noise ratio is around 6 times higher at 278K than at 310K.
[ppm]
15
GLY -.
.~~~~~~~
.
[ppm]
15
N
110.00
N
- 110.00
.
II, 1
O
s. a
.2.....
LA:
-
._
.--- ,
.
- 115.00
120.00
- 120.00
.-
o.
.'
.
-115.00
::
,eh
-:.
310-4 a _ .
^~~~~
P
VW-
A.
.:!
-125.00
't,~
-'--
310K
278K
I
I
9.00
.
...
I
.
8.50
.
125.00
a
.
.
J
.
8.00
,
.
.
.
.
.
7.50
.
.
.
.
-
.
7.00
1
.
.
9.00
.
.
.
.
I
8.50
....
I
I
I
I
8.00
I
7.50
1H
H [ppm]
'
'
7.00
[ppm]
Figure Z7:H, 5N HSQCspra qffie TCR (at 278K(lefi)and 310K(nit). Typia speatralrgiamfor sddcti arno
ads and the sidedain animog
a iated by gy shadig R msati monares tdt are akerMdat hi&r
tetrature am
reira
by armz. The spetm nat 278K hs beenobaird uth 4, the oneat 310 K uth 8 tramentsper tin
nEt
1D-slics hu a di reslud d
residuedwmtratethelasinsigml to nseratioathigber tneratue
157
Chapter4: Structure of the TCR C-chain
Despite the aforementioned disadvantages, early assignment experiments have been carried out at
310K in order to obtain a resonance assignment of free TCR ( at the same temperature as for TCR
4/LMPG, for which 310K is optimal (see below). Since the assignment could, however, not be
completed at higher temperature, additional measurements at 278K were carried out. Therefore, in
the assignment process, spectra at both temperatures have been used, which have been related by a
acquisition of a temperature series of H,1 5N-HSQC spectra in recorded in intervals of 1 to 2 degrees
and tracing of the 1H and
15N
shifts of every resonance.
The extremely small dispersion of only around lppm in the H dimension (Figure 7) lends further
proof to the assumed physiologically unfolded state of TCR 4. While the narrow chemical shift range
enhances resonance overlap, the unstructured state is advantageous for the assignment, since random
coil chemical shifts can be exploited. Accordingly, certain amino acids such as glycine, serine and
threonine and alanine are resolved from the other amino acids already in the 'H, 5N- HSQC (Figure
7). This tentative assignment however, is only adduced in support of the backbone assignment based
on a 3D-HNCACB, a 3D-HNCOCO and a 3D-HCCCONH (Figure 5).
[ppm]
13
c
20
30
40
50
60
9
hzawebeen
Figure : Stps fitn the 3DHNCA CB offiwe TCR 4at 278K For residues72 to 79 the HN-C ps
by
the basis of the assigmnt strategyin dhisspae,'nare a
extraa d The sa ontidi ard i-1 peaks, ubi& atnst
horizl
bas. Intraresidualresonancs are la by gr lettens.
158
Chapter4: Structure of the TCR C-chain
Figure 8 shows slices of the
N-C planes of residues 72 to 79 in the HNCACB. At the position of
every N-H moiety, the respective C~ and C_ resonances as well as the ones of the preceding residue
are visible. CQ,and Co resonances can be distinguished by their opposite sign.
The HNCACB sequence stretch depicted in Figure 8 can be readily assigned to residues 72 to 79
due to the presence of two alanines, which are one residue apart from each other, followed by a
serine and a glycine at a distance of one and four residue(s), respectively. In contrast, especially for
regions with the same or similar amino acids, the amino acid type sensitivity of the HNCACB
renders it insufficient for an unambiguous assignment. Thus, due to resonance overlap, residues 31
to 35 (sequence RREEY) do not show any sequential peaks in the HNCACB (Figure 9, left).
HNCACB
.
._
t,
P..,
p5.
.:,..
HCCCONH
HNCOCO
._
[ppm]
(ppm]
13c
13
[ppm]
c
Il
-20
2.0
i,89f+
7.1 - 30
.9
170
0
C
40
3.0
CP
si
171
-50
a a
-a
1H
I
yR
4.0
-60
R R E
31
a..__
-
E Y
-_--
172
35
31
.3 z
Jo l
M7a
Figure 9: StrPsfin t HN-Cplanesef dx 3D-MNCA CB (left), dhe
3DHN(
(idde) and thdx
3DHCCCXNH
ffi 7CR ¢ TYlxawtks in d HNCA CB and de HNCOX arela1w by gk lett'for dx alit ca, ,m
andpntormand"C "forthe as.
7x spinssten
the m peapran
w addas odad i theHCCCONH
am amradl by datedIis and kddai byge lette The maId "znai5
in theHNCXX) are iad
by solid
1
ls. Thepsitzon f themissingC reswm'of
residue Y35 in the HN4CACB is laid by a drde and t
nurks. TheHNCA CB hasbeendaid at 278K theHN(C
and theHCCCONHhaw bheen
rewda at 310K
159
Chapter 4: Structure of the TCR C-chain
In addition, in the case of residue Y35, the resonances are too weak to be detected. The same
(Figure 9, middle), which
sequence stretch can be easily assigned with the help of a 3D-HNCOCO
connects the carbonyl resonance of a residue with the carbonyl of both the preceding and the
following residue (Figure 5). In contrast to the C and C4 chemical shift, the C is much less amino
acid specific. Apart from that, the connectivity in the HNCOCO is established through different
scalar coupling) resulting in
scalar coupling) than in the HNCACB (C-C
interactions (C-C
different transfer efficiencies. Therefore, while residue Y35, which does not show cross peaks in the
HNCACB, it can be sequentially assigned in the HNCOCO. Additional certainty about the amino
(Figure 5), in which the complete carbon spin-
acid type can be received from the 3D-HCCCONH
system of the preceding amino acid is obtained at the position of every NH moiety (Figure 9, right).
With the sequential information contained in the three aforementioned spectra, the backbone
resonances (H, N, C, C) and the Cp of all but three residues of free TCR 4-chain could be
assigned at 278K (Figure 10).
ITAM1
A.......
ITAM2
.......
..........
'
.
.
10
..
'_
_-
'
1
'
.
.
.
''''I.' .......
.
30
20
I
40
50
ITAM3
_
Muzzan"a
80
80
70
I
90
110
100
[ppm]
G88
G49
is N
G50
G30
G91
G95
G81
-G43
;&G60
-110
G19 G86
,,~G78
I
I
8.5
I
I
I
8.0
8 .0
F
-
T
I
I
I
7.5
H [ppm]
Figurwe10: Resonaneassigmrnt offie TCR ; at 278K Thegry shad resides in the sepw
by a grey
im b
wRned
rsdai
Aztwi shading
12dd add nt eyassz
Reosnau assigmntsfbr
e 12 nowtemisn1
areshozmin an ZH,15N-HSQC
160
hae baeen
assig,
tentratwy asti
do
Chapter4: Structure of the TCR -chain
N63 was tentatively assigned, while E64 and L27 could not be assigned at all, since the resonances
were missing from the spectra. The assignment hence is complete in the sense that all the assignable
resonances have been also assigned, while residues that could not be assigned, do not show
resonances of sufficient sensitivity in the spectra. At 310K, the assignment is less complete, since a
number of resonances are significantly weakened. The backbone assignment at both temperatures is
summarized in the Appendix (Section A4; Table A2&A3). Figure 10 shows the assignment of the 12
non-terminal glycines in the upfield region of the
H,1 5 N-HSQC, demonstrating that also in this
comparatively resolved region resonance overlap is severe in unfolded TCR C.
For residues R4 to K6 and the Gterminal R115, a second set of resonances of minor intensity has
been identified (Figure 11), indicating the presence of two different conformations for the two
termini. While the two resonances for R115 can be readily explained by cis-trans isomerization of the
adjacent proline P114, the two sets of resonances in the N-terminal region are proof for the presence
of non-random-coil structure in this region, because at least one set of resonances has to arise from a
structural arrangement different from random coil. The existence of two sets of resonances for this
region demonstrates that the conversion between the two conformations either takes place on a slow
time-scale or they represent two species of TCR ~ altogether. The two conformations are present
over the whole temperature range (Figure 11) and exhibit about the same relative orientation
[ppm]
[ppm]
sN
F7
15N
OO
122
123
123
L3
0
O~
124
o~~
124
O°1
K6O~o
00*4O
125
K62
125
RiIS
126
278K
.II.
.
8.4
126
R1152
..........
.
8.2
.
.
8.0
.
.
.
7.8
'H [ppm]
8.6
8.4
8.2
8.0
7.8
'H [ppm]
Figure 11:SeaMionvmte 'H,'NHSQC f CR at 278K(left)and310K (it). Theto setsof rsona forR4K6andRl 5are laed Thes i esnm of L3 andF7amalso imt A t 310K the onanme
of L3, 'vbid appears
atlouer hs5
is idicated by an asterisk.
161
Chapter 4: Structure of the TCR C-chain
4.3.2.2
/Nef-interaction
The residues of TCR ~ involved in Nef-binding have been determined by adding unlabeled Nef to
5 N labeled
TCR ~ and comparison of the 'H
5N-HSQC
of the bound and free TCR 4-chain (Figure
12, above). While most of the resonances remain at the same position, some are shifted upon Nef
binding to a varying degree. This demonstrates that Nef is binding to the ~-chain.
In the Nef-bound spectrum of TCR C, a number of resonances have shifted to an extent that they
cannot be related to a resonance in the free state (Figure 12, resonances labeled by an asterisk).
Unfortunately, the identity of these residues cannot be elucidated since no assignment of TCR
bound to Nef is available. The quantitative interpretation of chemical shift changes as indicators for
the strength of an interaction may however be misleading in any case, since chemical shift changes
reflect alterations in the chemical environment, the extent of which might not be correlated directly
to the extent of involvement in a binding event. In case of an unfolded protein like TCR C, the
correlation is, however, more direct than for a folded molecule, where internal conformational
rearrangements
might complicate the identification of an interaction site. Based on a semi-
quantitative chemical shift analysis (Figure 12, below), two interaction sites of TCR 4 with Nef can
be identified, residues 4 to 15 and 51 to 58. A third stretch of minor chemical shift changes is located
at residue positions 41 through 43. In addition, small chemical shift changes are present along almost
the whole sequence. For residues V5 and K6 both sets of resonances exhibit the same extent of peak
shifts. Due to spectral overlap, resonance shift upon binding cannot be followed for the second
conformation of R4. Most of the observable residues (all residues besides prolines, which cannot be
detected in a H',N-HSQC,
since they do not possess an amide proton) within the two major
interaction regions exhibit resonance displacements to a degree that the resonance in the Nef-bound
state is so far shifted from the free form that it could not be identified. These resonances are
assigned in the 1 H, 5N-HSQC (Figure 12, above).
162
Chapter 4: Structure of the TCR 4-chain
l
Cm
C
[ppm]
5N
000
110
AM
115
S
gr
D12
a
a
40*
S
i
m
*n
N
el
120
K55;
aw
*
Amb
All
A13
,-Vm
K6
4
-_ ;
Vt!
*
*'D
alwt
125
A
m
to
H [ppm]
8.50
ITAM1
.ITAM2
ITAM3
di
C
0
x
0
0
U)
Ca
b-
ala
I ILbLngLjiLna
20
40
60
Residue
A 03
o
80
Figure 12: Caption cat on the rnext
paE
163
100
dL
Chapter 4: Structure of the TCR 4-chain
Figure 12 (pl vious page):
to uwad
/Nfineraction A hbe 1H, 5N-HSQCglffieeTCR (auk) and 15N-lah
Nef(gry) at 293K A 2:1 excessofNef has beenused at 0.2 nM TCR
dAnial shift fm
ro
indfie
m
between the o states,are assigec ew
state,a itiatd byasks.
leering in theyaxis signifihedfiula
owdappt nvmetan hlf
Nef-bwrl
fr
onrnepcssiW.d: ntoap
esid
, fuhiab noshft
Belowsen-qn
i d
. Resaym,
ich exhibit extemiiw
xr state, vz
caoyx be dated to
sbft
oti
qmn
Nefbiaig The
shiftsbetueenthe maunms ffim ;and 4At=7dto Nef a:
extnt ofrese
el b: owda
reswa in the Nef
g ls dan hdf cowArfc: notoziappungdse tier,
far aatt,nti
d lfeid ersitx
b
4dbad
d
f theNyn
t
on ,
notpsi
Iide
can ofbA
,o barisgiw
-spes ,ote at dx sanrpitiomorte ,d e isaprdim
These results demonstrate unambiguouslythat Nef binds to the cytoplasmicdomain of the T-cell
receptor C-chain.Furthermore, the interaction is restricted to two distinct regions of TCR C, while
the rest of
remains unperturbed, indicating specific, localized binding rather than an overall
structural transition. With all care with respect to the interpretation of the NMR data, it has to be
noted that neither of the three ITAM sequences is involved in the interaction. This disagrees with
previous biochemical data published in the Reinhart-group [3] (Figure 13). In addition, no elevated
chemical shift changes are detected in the region of either binding site determined in the mutagenesis
study.
Several explanations can account for the deviating results of the NMR and the mutagenesis study.
The observed differences could arise from the comparison of two different techniques, which
observe different parameters as probes for binding. The mutagenesis study is based on the detection
of binding events in a yeast-two hybrid assay [3]. In this in zw assay, it is possible that a number of
events could impair the 4/Nef-interaction such as alternate interactions or steric considerations.
4
15
51
N
.ITA.-A
24
58
I
01131111111011PW
.~
ITM21.T
2:.
34
74
;1 :.AM?3: t C
79
Figuw 1L3Nef btig sitesof TCR Thetwo jor interaeions
kts doby the NMR imiestion areshonmate
thesmzutic senpm Theanr add squaes aregm rtesides,for h signifant dnrical shiftdAr addbe idef
qp Nefbing amhilid
7heNfi eractionsitentfeier
164
are dicated owthesenm&
Chapter 4: Structure of the TCR C-chain
On the NMR side, the aforementioned possible indirect nature of NMR chemical shift
perturbations can result in differences between the detected and the actual binding site in these
experiments, especially since only the backbone amides have been investigated, while no information
on the side chains could be obtained.
Thus, Nef binding at the positions identified within the mutagenesis study might not be
accompanied by changes of the chemical environment to an extent to which secondary
conformational changes affect the regions identified by the NMR analysis. While this constitutes a
possible explanation for the absence of chemical shift changes in putative binding regions, regions
exhibiting chemical shift changes are still involved in the binding event.
A different explanation could be found that although both studies focus on the TCR ~ cytoplasmic
domain, the chemical shift perturbation study has been carried out on the isolated cytoplasmic
domain in solution (with three nucleotides of the transmembrane domain added to the N-terminus).
It is questionable if the interaction of Nef with the N-terminus of the TCR ~ cytoplasmicdomain is
biologically relevant as identified in the NMR-study, since this interaction would be most likely by
the presence of the cell membrane sterically complicated under physiological conditions. Hence, the
sterical accessibility of the N-terminus in our study could offer an additional non-physiological
binding site for Nef. Although both the TCR ~ cytoplasmic domain and Nef have been studied as
fusion proteins in the mutagenesis study, the presence of an additional protein at the N-terminus
should not interfere with Nef binding at the N-terminus since the two components of the fusion
protein are not directly attached to each other but separated by a putatively unstructured linker
region. It is possible, however, that apart from steric considerations, conformational freedom is
crucial for Nef binding to the N-terminus in the NMR study. On the other hand, it could also be that
involvement of both TCR 4 and Nef in fusion proteins causes changes in Nef binding from the
physiological situation.
Apart from this explanations, the differences in the Nef binding sites can also arise from the
intrinsic propensity of TCR ~ to dimerize, which has been reported only recently by our co-workers
[5]. Gel filtration and dynamic light scattering experiments support that the TCR
cytoplasmic
domain is present as a dimer at the concentration and temperature used in the Nef binding study. So
far, it is unknown whether Nef binds to TCR ~ in its monomeric or dimeric form. The strength of
the ;/Nef-interaction
(KD=0.3-2pM) is slightly higher as the one reported for the C/ interaction
165
Chapter4: Structure of the TCR 4-chain
(KD-10LM), indicatingthat disruption of the ,/ dimer by binding of Nef to the monomeric species
can take place at the concentrations studied (twofold excess of Nef). If Nef binds to the monomer,
chemical shift perturbations are expected both within the dimerization interface and within the Nef
interaction sites. Even if Nef is binding to the
/~-dimer, the observed chemical shift perturbations
could arise from conformational changes within the dimer upon Nef binding as well as from direct
interactions with Nef.
4.3.2.3 q/-Dimerization
In order to dissect possible monomer-dimer transitions from Nef binding, both the C/4-dimer and
the C-monomer need to be NMR-spectroscopically characterized at the conditions of the ~/Nefinteraction study (293K, pH7). A first attempt to achieve this has been a concentration dependent
chemical shift analysis. In the presence of 100mM NaC1 as it is the case in the ~/Nef-binding study,
the KD of the 4/4-interaction amounts to about -10[tM [5]. These conditions are unfortunately well
beneath the concentrations that can be studied by NMR. However, according to our co-workers, the
KD of C/4-dimerization increases to around 3001 tM at lower salt conditions. Therefore,
'-L 5N-
HSQC spectra at 400pM and 10[iM of TCR 4 under low salt conditions (no NaCL was added to the
sample) were investigated for chemical shift changes (Figure 14). In general, only very slight
deviations in the chemical shifts could be detected in these experiments. In particular, the 'H
chemical shift changes are so minute (on average 0.003 +0.00lppm) that they have been disregarded
in the further analysis. From the analysis of l5N chemical shift perturbations, only the region between
residue 24 and 42 exhibits concerted changes (Ab-S -0.08 ppm for seven out of the 19 residues).
This region is, however, far away from the interaction sites with Nef. Of the two single residues with
elevated chemical shift changes, R4 and Q94, the former one constitutes a part of the binding
interface to Nef. It is remarkable that all the resonances of the second conformation of residues R4
to K6 exhibit large concentration dependent chemical shift changes (Figure 14).
Apart from the concentration dependence, the monomer-dimer equilibrium is also dependent on
temperature.
Therefore, the temperature dependent 'Hl 5N-HSQC series used to transfer the
assignment at 278K to the one at 310K has been also analyzed for the extent of chemical shift
changes (Figure 15). The observed temperature dependent chemical shift changes are about one
order of magnitude higher for "5N and two orders higher for 1H than the concentration dependent
ones. Therefore, both nuclei are considered in the chemical shift analysis in this case.
166
Chapter 4: Structure of the TCR -chain
[ppm]
4a
OW
15N
112
T100 *
_
114
a0
116
o
o *:
S10 ~ e
118
R41a
R 4 l-%,.h
Y24
a
,_l"a-,N25
_ - _*
~~ F
a
W4 ll
L3
v
a
© ['
L62
~L61.
D39R42
e
R41-
11
*S
MM9L624R115*
R11
R115
~
a
_
400 tM
122
124
'0
a
120
126
i
I
8.4
8.6
W
1H
ITAM2
ITAM1
0.15 .0
m
8.2
[ppm]
ITAM3
0.1-
z
(O
0.05
-l
-
020
40
60
Residue
80
100
4-6
2"~ Conf.
at 400pM (badk) and
di ntrizationstudiesqfthe -dain. H,l5N-HSQC spectra
14: Conntration
(ubs)
as pltd againstthe
cl shiftdf
10OpM
(grey);-aweration at 293K are ozwlaL A bsolute15N
Figur
reide m
c
v
bow the spectn
Theputatze dn
zation site i ndicataed
by greyshadirg. CQeia sht dhans for the
by
hig esabi
than 005 ppm a la
secand set of resonancrsf residue4 to 6 are also gizen Residuh
arrw. Peaks, vh
appearat lozer co ration, are indicatedby asteriks; shiftingresdu am assigr
167
Chapter4: Structure of the TCR -chain
- [ppm]
15
G95
- _
..
N
G78
I
_1
1
-112
W
....
"JIA-.
_r
T03
,,
-
_
-114
T100 310K
T98
8.2
8.0
8.4
8. 4
278K
I
HI ppm]
ppm]
I[
:..
1
(a
co
&Z
0.5
0
WV
.
., .¢
-.
'.'
ITAMI !:.-:^
i.
0.2
D
'_
;~~
:,
.,
,.
'
'.:
i.:
:
0
-'
"-,
I..;'
11
20
d
w1
_...11:
.111111
60
40
dim
"":i:~-
triznstdi
f te 4-dh.
.
.
I
....
..
.
I
,i '' . '',
. '., I:.
.
'.
I
:.' ". :.,
'::
-
!: '
ll1:
."l 11|.
w 11: w: :11 1 w111 w: w
80
100
Residue
Figuw 15: Tpramre
'
':,
.
< 0.1
..
-:/
1IM2
_
,.,...
..
'.
....'.'
., '.!.,-:v
:
4-6
2 nd
The
e
Conf.
and so/,rediem
mg q IH,15N
residuff are assid
HSQC speara at 278, 283, 293 and 310K (Hade to lightgmey)are ozeda)
Resdzlid exera
the checal shift dmngsuponteqratue ire
H and 15N dreical shift dn
spertra
at 278 and 310K (Aoa
are idicateduith arr.
bs)
and
kzen the
15Ndxnmiadshiftd
and
(ab(A H - nAcean))
ibdow
d speno
N~~~
// arepki agiimsttheresidemir
s amplktd as absdlutewes. A eragedx l shf dms am
reidiated by dd hozvtld I.
Reo'
ted highdxcniashif
echibiti
dh
am higlited
168
Chapter 4: Structure of the TCR C-chain
While "5 N resonances show both positive and negative chemical shift changes upon increase of
temperature, the imino-protons exhibit concerted downfield shifts. This is due to their dependence
on the exchange rate with the solvent water in addition to their conformational dependence.
Accordingly, the total H chemical shift changes have been not been interpreted directly but the
absolute deviation from the mean chemical shift changes have been considered as a measure for
conformational changes. In addition, proton chemical shift perturbations are evaluated only in
support of the "5N data or in cases of extreme deviations. Four regions with elevated "5 N chemical
shift changes are observed (residues 4-10, 16-17, 37-40 and 100-108); a fith putative dimerization
region can be identified at sequence positions 93 to 95 based on an extreme H chemical shift change
of 0.5 ppm for G95 and elevated "5 N chemical shift changes for Q94 and Y93. While the regions
between residues 37 and 40 and 93 to 94 coincide with the putative dimerization site identified bythe
concentration dependent chemical shift changes, the N-terminal region (4-10) constitutes a part of
the identified Nef binding site. In contrast, the region between residue 100 and 108 has not been
detected in either other study.
4.3.3 TCR Cbound to LMPG-micelles
Prior to the structural investigation of the LMPG-bound TCR (, the :LMPG ratio was optimized
by analysis of the resolution and sensitivity in H,'5 N-HSQC spectra with protein:detergent ratios
between 1:150 and 1:600. A 300fold excess of LMPG to TCR ; was found to be optimal at a 0.4
mM concentration of the TCR ( (data not shown). Temperature optimization in the same fashion
yielded 310K as optimal. As demonstrated in (Figure 16, left) for the glycine region at 310K and
318K, the sensitivity is significantly decreased at temperatures higher than 310K due to more
dramatic line-broadening. At lower temperatures (data not shown), sensitivity goes down as well.
Higher temperatures are favorable due to a decrease in viscosity resulting in line-narrowing. At
temperatures higher than 310K, however, the benefit of lower viscosity is counteracted by another
effect, possibly thermal disruption of the detergent micellesor increased exchange processes of TCR
C, leading to increased line-broadening. Therefore, the structural investigation was carried out at
310K.
The 1 H, 5 N-HSQC of TCR ~ in the presence of LMPmG-icelles compared to free TCR ~ exhibits
strongly increased line-widths (Figure 16, ight) at only slightly increased spectral dispersion, leading
to a dramatic decrease in resolution.
169
Chapter 4: Structure of the TCR -chain
[ppm]
310K
N
-100
08
%,
..
( ppm]
+ LMPG
free
N
~~~~~~~~~~110.00
,~~~~~~~~~~~~.
am109.00
-110.00
8.60
318K
8.40
115.00
8.20
'H ppm]
318K
>,,r .
[ppm]oX~s
N
120.00
108. 00
109.00
.
125.00
-110.00
i
8.60
l l l l l l ,I ,.,,,,,,.,, ,,, I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~,,
i
8.40
i
i
i
,
. . . . . . . . . . . . ....i..
9.00
8.20
H [ppm]
8.50
8.00
7.50
7.00
6.50
'H ppm]
Figure 16: 7TCR bwd to LMPG ridla. Left G*wd gin of H,5N-HSQC spera of LMPGbkma TCR at
31 K(top)and 318K(bttar). Right 'H,1 5N-HSQCspera cffr 7CR (iac) and 7R
boid to LMPG nidles
(gry) at 310K
The similarity in resonance positions between free and micelle-bound TCR ~ is minimal, signifying
an overall structural rearrangement upon micelle-interaction.
The backbone assignment of detergent-bound TCR ~ has been based on a 3D-HNCACB and a
3D-HNCACO. Residue type specific assignments were extracted from a 3D-HCCONH
and a 3D-
HCCCONH. The two partly redundant TOCSY experiments were obtained for the subsequently
planned side-chain assignment. The less informative 3D-HNCAGO (Figure 5) had to replace the 3DHNCOCO in this case, since the association with the detergent micelle increased the relaxation of
TCR ~ and the longer 3D-HNCOQ
sequence could no longer be carried out with sufficient
sensitivity.
The resonances show widely varying intensities in the spectra. Thus, several stretches could be
readily assigned from the connective information of the HNCACB and the HNCAQO alone, as
shown for residues 33 to 41 in Figure 17 to the left.
170
Chapter 4: Structure of the TCR 4-chain
HNCACB
HNCACB
[ppm]
13
HCCCONH
...
I
P..P .
C
-.........
[ppm]
J1
.tIb:PA#Zt
13
-
':3''#
40.00
*444'!1k?
-
40.00
cc
OC"'%~
- 50.00
am
4z
Is
...a
-9
-4
HC
HX
50.00
OC
P.
- 60.00
E E y D V
33
..
D K R
Cx
H
41
G L
D
Y
88
Q
[ppm]
[ppm]
13
13 c
C
SC' 'f H 'D G YQI
175.00
"
;~i,-
-- O
HDGLYQ~°
D
EEYDVLDKR0
94
HNCACO
HNCACO
33
- 60.00
1
41
~
88
175.00
176.00
94
Figure 17: Spectrausedfor the assignmnt qf TCR bmid to LMPG. Left Stripsforn e HN-C plane of reside 33 to
41 fromthe 3D-HNCA CB (top)
and the 3D-HNCA CO (btt;
i to i-1 mmotiies are inicated by horizornalbas.
Right. Owa
stips from the hN-C planes of residue89 to 94 of the 3D-HNCA CB (ba contcars)and the 3DHCCCONH (greyoonts) (t) and the 3D--HNCA
(kbon); i to iiities uithin the HNCA CB ae
indicated by dotted lis,
c
thoe that
dd only he established based on the HCCCCONH
are gizen by solid lines. The I to i- 1
zmctiuty
in the HNCA CO is indicatedby a sdolidline
In contrast, in other regions, both the HNCACB and to an even higher degree the HNCACO
show only the strongly weakened intraresidual peaks (Figure 17, right), with i-1 peaks that, if they are
present at all, are only visible upon meticulous analysis of the respective 1-D slices. In these cases,
mainly the strong C'
resonances in the HCCCONH
have been used to establish connectivity,
which were then verified in the HNCACB. This strategy is demonstrated in the right part of Figure
17, where strips from the HNCACB and the HCCCONH are overlayed.
In addition to stretches of weak resonances in the assignment, a number of resonances could not
be assigned or were missing from the spectra entirely. As a consequence, 14 residues are missing
entirely from the final backbone assignment (Figure 18) and an additional 5 residues could only be
tentatively assigned. The assignment is summarized in the Appendix (Section A4; Table A4).
171
Chapter 4: Structure of the TCR C-chain
ITAM1
GR
"
.
.
.
ITAM3
ITAM2
70
Figure 18: Badek
60
50
40
30
20
10
80
resane
100
90
assignmrntq t he TCUR bKoz to LMPG-i.
assig' wdediw esidues not id lyassig' tmaedy assiresi
110
The residu shadl in grey dld te
s are la
gh ey aces.
The chemical shift indices (CSI) for H, chemical shift perturbations from random coil chemical
shifts [30] have been used to investigate the presence of secondary structure within the assigned
regions of LMPG-bound TCR ~ as reported from CD-experiments [1]. While regions of at least 4
consecutive residues exhibiting a CSI of +1 are indicative for 3-sheet conformation, stretches with a
CSI of -1 report on an a-helical arrangement [30]. For the regions of LMPG-bound TCR C, for
which a HL chemical shift could be obtained from the HCCOC)NH, no evidences for any secondary
structure can be observed (Figure 19). Therefore, it is concluded that the helical regions have to be
located within the areas of TCR C, which are unassignable in the NMR spectra due to extensive
resonance broadening, namely residues within or adjacent to the ITAM2 and ITAM3. These regions
also coincide with the longest stretches of predicted a-helical structure based on the primary
sequence as predicted by the program PSIpred. Therefore, it is proposed that the regions around
ITAM2 and ITAM3 adopt helical structure upon interaction with the lipid micelle
Sequence
ITAM1
ITAM2
ITAM
.S.'AYGMGERRRCKGHDLYQG-/ALHM2APPR
'N&
GSLRVKFSRSADAPAYQQGgNQLYNELNLGRREEYDVLDRRGRDPEM(KGKPRRKNPQEGLYN
1'0
20
30
40
50
60
70
do
go
100
110
Prediction
Chemical shift Index
3?111H
01
-1
1 11"
L|
l
I I
fthe aws sible gm fLMPGbwd TCR 4, thesemnarystnamepw tdbyPSIpred based
Figure 19:Alit
on d aino acidsauere and CSI aniis of dxeHa dial shifts.Ha dxnil shiftshae beenextratedfian dx 39
HCCCNH spean Next reibor dependt randomai dxnial shiftshae beenobtaid fro theprogramNMRuew
+0.1ppmnrults in a
dxnial shif firn therandomail due er thadn
detiaim of dxeecxit
[31, 32]. Positiwe
of eier sign,the CSI is 0.
mrthan-0.ppmrsul ina CSI of -1,forsnerdeiat
CSI f +1, regaiw deiat i
etem
elHadnicd shot bysmll ban.
fiantede
Rfidues uith a CSI q0 hazebeen di
172
Chapter 4: Structure of the TCR 4-chain
Since the parts of the protein, containing two of the three interesting ITAM motifs could not be
assigned and the Ha chemical shift analysis does not indicate the presence of secondary structure
elements within the assigned parts of the sequence, any further structural investigation on LMPIVGbound TCR ~ was abolished. The increase in line-width in or adjacent to ITAM2 and ITAM3 of
LMPG-bound TCR C, which impairs resonance assignment, however, carries structural information
on its onw. The line-width of a resonance is determined by its transversal relaxation rate, which is
sensitive on the one hand to slow exchange processes and on the other hand to fast molecular
reorientation
events.
Thus, an increase in line-width signifies the presence
of increased
conformational exchange or the involvement in a structural arrangement of increased molecular
reorientation times, e. g. micelle incorporation. The increased line-width of all resonances of TCR ;
in the presence of LMPG as compared to free TCR reflects involvement with the detergent micelles.
The additionally broadened resonances in the region of ITAM2 and ITAM3 allow for different
~/micelle interaction models (Figure 20):
The regions of TCR C,which exhibit increased line-broadening, could be stably associated with the
LMPG-micelle (Figure 20A). Consequently, these residues experience the increased reorientation
times of the micelle leading to line-broadening, while the remainder of TCR ~ is going in and out of
it or just moving freely in the solution. An alternative explanation is that the two ITAM regions are
exchanging between different lipid bound forms (Figure 20B) or a lipid bound and a free form
(Figure 20Q), thus causing exchange broadening of the concerned resonances, whereas the remainder
of TCR ~ protrudes freely into solution. The first hypothesis is unlikely, considering that extremely
modest changes in relaxation behavior have been reported for proteins of the size of TCR ; when
incorporated into LMPG-micelles [33]. In this earlier investigation, the micelle bound form of a 12
kDa protein showed the relaxation behavior of a 20 kDa molecule and exhibited correspondingly
narrow line-widths. It is reasonable to assume that C, which possesses a molecular weight of around
13 kDa, exhibits a similar relaxation behavior. Therefore, a conformational exchange model for the
TCR
/LMPG-micelle
interaction
(Figure 20) is more
likely. While the conformational
rearrangement on the LMPG-micelle (Figure 20B) would not necessarily be accompanied by changes
in the TCR 4 secondary structure, the exchange between a micelle-bound and a free form (Figure
20,C) includes changes from a helical to a random coil structure in the regions of ITAM2 and
ITAM3.
173
Chapter 4: Structure of the TCR 4-chain
A
B
_
is
ij' -p
-
)t
.- 00
A-40-
N
Figure 20:Modiesqf
T/LM
PGnicde
eraaid
4.4 CONCLUSIONS
In the course of this work, the backbone assignmentof both the free TCR ~ and TCR 4 bound to
LMPG-micelles has been achieved. The almost complete assignment of the free form provides an
NMR spectroscopic fingerprint, which constitutes the outset of a residue specific investigation of the
binding site of SIV Nef. In order to identify this binding site, however, the TCR ~/Nef-interaction
sites have to be dissected from the ones of the C interaction. While the conditions, under which the
TCR 4/Nef-interaction
of a
studies as well as the assignment experiments were carried out, are in favor
/4-dimer, the 4 monomer has so far not been characterized NMR spectroscopically. First
concentration as well as temperature dependent investigations aimed at the identification of the C/4interaction sites as well as at characterization of the
monomeric species, have proven partly
incongruent. So far, only minute changes could be identified in the concentration dependent study,
while a number of putative interaction regions could be identified by the temperature dependent
experiments. Temperature dependent chemical shift changes can, however, arise from reasons other
174
Chapter 4: Structure of the TCR C-chain
than changes in the 4/-interaction, since the conformational ensemble defining the chemical shift of
a spin in an unfolded protein such as TCR ~ can change with temperature due to different energy
barriers for different conformational states within the ensemble. In the case of the imino protons,
solvent exchange influences the chemical shift in addition to conformational aspects. In addition, no
defined end point was reached at higher temperature in the investigated temperature region from
278K to 310K, signifying that the experiment failed to achieve conditions, under which the pure
monomer could be NMR spectroscopically characterized. From the unsuccessful concentration
dependent experiments, which have been carried out at the same temperature as the
/Nef-
interaction study, it seems to be impossible to obtain a spectrum of the monomer at these
conditions. Therefore, further studies will aim at a conclusive characterization of the 4/-interaction
site(s) by concentration dependent studies at higher temperatures, where the monomer-dimer
transition is shifted to higher concentrations.
Even under the optimized conditions in regard to the TCR (:LMPG ratio and the temperature, the
backbone assignment of TCR ( bound to LMPG-micellescould not be completed due to extensive
broadening of a number of resonances. Because of this incomplete assignment, the structural
investigation of LMPG-bound TCR ( was not carried any further than the backbone assignment.
Structural information could however still be obtained for both the assignableand the unassignable
regions. A chemical shift analysis measuring deviations from the random coil chemical shifts as an
indicator for the presence of secondary structure demonstrate that the assignable regions do neither
adopt a-helical nor P-sheet structure. Consequently,the detected a-helical content of TCR ( upon
micelle-binding has to be located within the non-assignable, broadened regions, located at the 2 nd and
3r
ITAM motif. This is in agreement with sequence-based
secondary structure predictions.
Furthermore, the increase in line-width in these regions indicates the involvement in chemical
exchange processes in the millisecond range. Different interaction models with the LMPG micelle
have been proposed here, which are in agreement with the localized occurrence of elevated slow
exchange (Figure 20). In conclusion, the study demonstrates that only the regions around ITAM2
and ITAM3 (and not ITAM1) can adopt a-helical structure. These regions are also the most likely
interaction sites with the LNPG micelle.
175
Chapter4: Structure of the TCR c-chain
4.5 REFERENCES
1.
Aivazian, D. and Stem, L. J. (2000). Phosphorylation of T cell receptor zeta is regulated by a
lipid dependent folding transition. Nat Seie Bid, 7, 1023-1026.
2.
Bell, I.; Ashman, Q; Maughan, J.; Hooker, E.; Cook, F.; Reinhart, T. A. (1998). Association
of simian immunodeficiency virus Nef with the T-cell receptor (TCR) zeta chain leads to
TCR down-modulation.J Gen Vini, 79 ( Pt 11),2717-2727.
3.
Schaefer, T. M.; Bell, I.; Fallert, B. A.; Reinhart, T. A. (2000). The T-cell receptor zeta chain
contains two homologous domains with which simian immunodeficiency virus Nef interacts
and mediates down-modulation.J Virol, 74, 3273-3283.
4.
Xu, X. N.; Laffert, B.; Screaton, G. R; Kraft, M.; Wolf, D.; Kolanus, W.; Mongkolsapay, J.;
McMichael, A. J.; Baur, A. S. (1999). Induction of Fas ligand expression by HIV involves the
interaction of Nef with the T cell receptor zeta chain.J Exp Med, 189, 1489-1496.
5.
Sigalov, A.; Aivazian, D.; Stem, L. (2004). Homooligomerization of the cytoplasmic domain
of the T cell receptor zeta chain and of other proteins containing the immunoreceptor
tyrosine-based activation motif. Bi/onstry,
43,2049-2061.
6.
Germain, R N. (1994). MI-HOdependent antigen processing and peptide presentation:
providing ligands for T lymphocyte activation. Ced, 76,287-299.
7.
Alberola-Ila, J.; Takaki, S.; Kemrner,J. D.; Perimutter, R. M. (1997). Differential signaling by
lymphocyte antigen receptors. A nnu Revimnrd, 15, 125-154.
8.
Qian, D.; Griswold-Prenner, I.; Rosner, M. R.;Fitch, F. W. (1993). Multiple components of
the T cell antigen receptor complex become tyrosine-phosphorylated upon activation. J Bid
Gbem 268, 4488-4493.
9.
Baniyash, M.; Garcia-Morales, P.; Luong, E.; Samelson, L. E.; Klausner, R D. (1988). The T
cell antigen receptor zeta chain is tyrosine phosphorylated upon activation. J Bid (em, 263,
18225-18230.
10.
Iwashima, M.; Irving, B. A.; van Oers, N. S.; Chan, A. C;Weiss, A. (1994). Sequential
interactions of the TCR with two distinct cytoplasmic tyrosine kinases. Sdoze, 263, 11361139.
11.
Hatada, M. H.; Lu, X; Laird, E. R; Green, J.; Morgenstemrn,J. P.; Lou, M.; Marr, C S.;
Phillips, T. B.; Ram, M. K.; Theriault, K.; et al. (1995). Molecular basis for interaction of the
protein tyrosine kinase ZAP-70 with the T-cell receptor. Natu, 377, 32-38.
12.
Samelson, L. E. (1999). Adaptor proteins and T-cell antigen receptor signaling. ProgBiqs
Md Bid, 71, 393-403.
13.
Clements, J. L. and Koretzky, G. A. (1999). Recent developments in lymphocyte activation:
linking kinases to downstream signaling events. J COinInest, 103, 925-929.
14.
Janeway, C A., Jr. (1995). Ligands for the T-cell receptor. hard times for avidity models.
Inwma
15.
Today, 16, 223-225.
Cochran, J. R; Aivazian, D.; Cameron, T. O.; Stem, L. J. (2001). Receptor clustering and
transmembrane signaling in T cells. Tmrls BiaodmSci, 26, 304-310.
176
Chapter 4: Structure of the TCR 4-chain
16.
Delon, J.; Gregoire, C; Malissen, B.; Darche, S.; Lemaitre, F.; Kourilsky, P.; Abastado, J. P.;
Trautmann, A. (1998). CD8 expression allows T cell signaling by monomeric peptide-MIHC
complexes. In
ity, 9, 467-473.
17.
Grakoui, A.; Bromley, S. K.; Sumen, C; Davis, M. M.; Shaw, A. S.; Allen, P. M.; Dustin, M.
L. (1999). The immunological synapse: a molecular machine controlling T cell activation.
Sie7e, 285, 221-227.
18.
Deacon, N. J.; Tsykin, A.; Solomon, A.; Smith, K.; Ludford-Menting, M.; Hooker, D. J.;
McPhee, D. A.; Greenway, A. L.; Ellett, A; Chaffield, C; et al. (1995). Genomic structure of
an attenuated quasi species of HIV- 1 from a blood transfusion donor and recipients. Sae,
270, 988-991.
19.
Salghetti, S.; Mariani, R.; Skowronski, J. (1995). Human immunodeficiency virus type 1 Nef
and p561ck protein-tyrosine kinase interact with a common element in CD4 cytoplasmic tail.
ProcNatldA cadSci USA, 92, 349-353.
20.
Baur, A. S.; Sass, G.; Laffert, B.; Willbold, D.; Cheng-Mayer, C.; Peterlin, B. M. (1997). The
N-terminus of Nef from HIV- 1/SIV associateswith a protein complex containing Lck and a
serine kinase. Inmoty,
6,283-291.
21.
Collette, Y.; Dutartre, H; Benziane, A.; Ramos, M.; Benarous, R.; Harris, M.; Olive, D.
(1996). Physical and functional interaction of Nef with Lck HIV-1 Nef-induced T-cell
signaling defects. J Bid
em, 271, 6333-6341.
22.
Lee, C. H; Leung, B.; Lemmon, M. A.; Zheng, J.; Cowburn, D.; Kuriyan, J.; Saksela, K.
(1995). A single amino acid in the SH3 domain of Hck determines its high affinity and
specificity in binding to HIV- 1 Nef protein. EnboJ, 14, 5006-5015.
23.
Saksela, K.; Cheng, G.; Baltimore, D. (1995). Proline-rich (PxxP) motifs in HIV-1 Nef bind
to SH3 domains of a subset of Src kinases and are required for the enhanced growth of Nef+
viruses but not for down-regulation of CD4. EnidJ, 14,484-491.
24.
Lu, X.; Wu, X.; Plemenitas, A.; Yu, H.; Sawai, E. T.; Abo, A.; Peterlin, B. MI.(1996). CDC42
and Racl are implicated in the activation of the Nef-associated kinase and replication of
HIV-1. Curr Bid, 6, 1677-1684.
25.
Greenway, A.; Azad, A.; Mills, J.; McPhee, D. (1996). Human immunodeficiencyvirus type 1
Nef binds directly to Lck and mitogen-activated protein kinase, inhibiting kinase activity. J
Vita, 70, 6701-6708.
26.
Sawai, E. T.; Baur, A; Struble, H; Peterlin, B. M.; Levy, J. A; Cheng-Mayer, C (1994).
Human immunodeficiency virus type 1 Nef associates with a cellular serine kinase in T
lymphocytes.Prc NadAcad Sci U S A, 91, 1539-1543.
27.
Smith, B. L.; Krushelnycky, B. W.; Mochly-Rosen, D.; Berg, P. (1996). The HIV Nef protein
associateswith protein kinase C theta. J Bid (xem 271, 16753-16757.
28.
Wittekind, M. and Mueller, L. (1993). HNCACB, a High-Sensitivity 3D NMR Experiment to
Correlate Amide-Proton and Nitrogen Resonances with the Alpha-Carbon and Beta-Carbon
Resonances in Proteins.JMagnReonSerB,
101, 201-205.
177
Chapter4: Structure of the TCR c-chain
29.
Montelione, G. T.; Lyons, B. A.; Emerson, S. D.; Tashiro, M. (1992). An Efficient Triple
Resonance Experiment Using Carbon-13 Isotropic Mixing for Determining SequenceSpecific Resonance Assignments of Isotopically-Enriched Proteins. J Am Cbem Soc, 114,
10974-10975.
30.
Wishart, D. S.; Sykes, B. D.; Richards, F. M. (1992). The chemical shift index: a fast and
simple method for the assignment of protein secondary structure through NMR
spectroscopy. B/xristry,
31.
31, 1647-1651.
Schwarzinger, S.; Kroon, G. J.; Foss, T. R.; Chung, J.; Wright, P. E.; Dyson, H. J. (2001).
Sequence-dependent correction of random coil NMR chemical shifts. J A m C/rn Soc, 123,
2970-2978.
32.
Wishart, D. S. and Sykes, B. D. (1994). The 3 C chemical-shift index: a simple method for the
identification of protein secondary structure using 13C chemical-shift data. J Bion NMR, 4,
171-180.
33.
Krueger-Koplin, R. D.; Sorgen, P. L.; Krueger-Koplin, S. T.; Rivera-Torres, I. 0.; Cahill, S.
M.; Hicks, D. B.; Grinius, L.; Krulwich, T. A.; Girvin, M E. (2004). An evaluation of
detergents for NMR structural studies of membrane proteins. J Bion NMR, 28, 43-57.
178
Appendix
APPENDIX
179
Appendix
TABLE OF CONTENTS
A1
CHAPTER 2: r(HCN)-Experiment
A1.1
......................................................................
181
Pulseprogram s ........................................................................................................................181
A l.1.1
F(H
.......................................................................................................................... 181
184....................................................
A1.1.2
r(H2'C',
.....................................A2.1
F-rate calculation ..................................................................................................................187
A1.1.3
Source-file ......................................................................................................................188
A1.1.4
Getrate
189
frompdb.sh ................................................................................................
A1.1.5
Get_ratefrompdb.c
..................................................................................................189
Coordinate file independent -rate calculation ........................................................191
A1.1.6
195
...............................................................................................................................
A 1.2 References
CHAPTER 3: RNA Tetraloop Dynamics.............................................................196
A2
A2.1 Pulse sequences ......................................................................................................................196
A2.1.1
RI .....................................................................................................................................196
A2.1.2
Rlp...................................
198
A2.1.3
"3CHetnOe ...............................................................................................................201
A2.2 Extraction of peak intensities ...............................................................................................202
A2.2.1
Autopkhpos.mac ...........................................................................................................202
A3
GHAPTER 4: Structum of the TCR c-chain ........................................................ 203
A3.1
Assignment of free TCR
at 278K ....................................................................................203
A3.2
Assignment of free TCR at 317K ....................................................................................
205
A3.3
Assignment of TCR /LMPG at 310K ..............................................................................207
180
Appendix
A1 CHAPTER 2: F(HCN)-Experiment
Al.l Pulseprograms
AI.1.l
F(HCN)
A1.1.1.1 Reference Experiment
;ghcn_final_ref
DELTA5
(center (p2 phl) (p22 phl):f3)
DELTA6
pl9:gp6
d16
4u plO:f2
4u
(center (pl4:sp3 phll):f2
(p22 phl2):f3)
4u
#include <Avance.incl>
#include <Grad.incl>
#include <Delay.incl>
"p2=pl*2"
"p4=p3*2"
"p22=p21*2"
"dO=3u"
"dll=30m"
"dl3=4u"
4u p2:f2
pl9 :gp6
d16
DELTA6
(center (p2 phl) (p22 phl):f3)
DELTA5
(p21 phl6):f3
4u
(p3 phl5):f2
d13
pl6 :gp7
d16
(p2 phl):fl
(p3 phl8):f2
dO
4u plO:f2
(center (p29:sp2 phl) :f2 (p2 phl) :fl)
DELTA2
p16 :gp3*EA*-l
d16 plO:f3
d13
(center (pl4:sp3 phl):f2
(p30:sp9 phl):f3)
d13
"in2O=in0"
"d6=d4-p8/2-4u"
"d20=d22-d13-p16-d16-p29-12u"
"DELTA1=d22-p16-dli6"
"DELTA2=d22-d16-pl6-dO-4u-p29-dl3"
"DELTA3=dl3+pl6+dl6+4u"
"DELTA5=d26/4"
"DELTA6=d26/4-p19-d16-8u"
1 ze
2 dll do:f2 do:f3
dl
(pl phi)
4u
d6 plO:f2 p13:f3
(center (p2 phi)
d6 UNBLKGRAD
4u
p28 phi
(p8:spl3 phi):f2)
dl3 p2:f2
pl6:gp3*EA
(pl ph2)
d13
pi6:gpl
d16
(p3 ph3):f2
DELTA1
pl6:gp2
d16 plO:f2 plO:f3
(center (pl4:sp3 phll):f2
(p30:sp9 phl2):f3)
DELTA1
pl6:gp2
d16
d20
(p29:sp2 phl):f2
4u
4u pl2:f2
4u
(center (pl phl)
d4
2
(center (p phl)
d4
(center (pl ph2)
4u
d16 p2:f2 p3:f3
d6 pO:f2
(p3 phl3):f2
d13
pl6:gp5
d16
(p3 phl4):f2
4u
(p21 phl7):f3
(center (p2 phl)
d6
4u
(pl phl)
DELTA3
(p2 phi)
d13
;ZQ/DQ-Delay
181
; Bloch-Siegert
(p3 ph8):f2
(p4 phl):f2
(p3 ph9):f2
(p8:spl3 phl):f2)
Appendix
p16:gp4
d16 pll2:f2 pll6:f3
4u BLKGRAD
go=2 ph31 cpd2:f2 cpd3:f3
dll do:f2 do:f3 mc #0 to 2
F1EA(igrad EA & ip9*2, idO & dd20 &
ip6*2 & ip18*2 & ip3l*2)
exit
phl=0
ph2=1
ph3=0
ph8=0
ph9=3
0 0 0 2 2 2 2
0 2 2
3 1 1
phll=0
phl2=0
ph13=1
1 1 1 1 1 1 1 3 3 3 3 3 3 3 3
;c13 1. puls;
ph14=0
;c13 2. puls;
phl5=
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2
;c13 3. x for ref, y for cross
ph16=0 2 ;n15 2. x for ref, y for
cross
ph17=0
;n15 1.puls;
phl8=1
;c13 after chem. shift
evolution ;y for ref, x for cross
ph31=0
2 2 0 2 0 0 2
2 0 0 2 0 2 2 0
2 0 0 2 0 2 2 0
0 2 2 0 2 0 0 2
182
Appendix
A1.1.1.2 Cross Experiment
(p22 phi2) :f3)
4u
;ghcn_final cross
#include
#include
#include
4u p2:f2
<Avance.incl>
<Grad.incl>
<Delay.incl>
p19:gp6
d16
DELTA6
(center (p2 phl) (p22 phl):f3)
DELTA5
(p21 phl6):f3
4u
(p3 phlS) :f2
d13
p16:gp7
d16
(p2 phl):fl
(p3 phl8):f2
dO
4u plO:f2
(p2 9:sp2 phl):f2
d5
(p2 phl):fl
DELTA2
"p2=pl*2"
"Ip4=p3*2"
"p22=p21*2"
"dO=3u"
"dll=30m"
"d13=4u"
"in20=inO"
"d5=d4-p29"
"d6=d4-p8/2-4u"
"d20=d22-d13-p16-d16-p29-12u"
"DELTA1=d22-p16-dl6"
"DELTA2=d22-d19-p16-dO-p2-p29-d5-4u
p16:gp3*EA*-l
-d13"
"DELTA3=dl3+p16+dl6+4u"
"DELTA5=d26/4"
"DELTA6=d26/4-p19-d16-8u"
d16 plO:f3
d13
(center (p14:sp3 phl):f2
1 ze
d13
2 dll do:f2 do:f3
p16:gp3*EA
(p30:sp 9 phl):f3)
dl
d16
d20
(p29:sp2 phl):f2
4u
(pl phi)
4u
d6 plO:f2 p3:f3
2
(center (p phl) (p8:spl3 phl):f2)
d6 UNBLKGRAD
4u
p28 phl
4u p2:f2
4u
(center (pl phi) (p3 ph8):f2 )
d4
(center (p2 phi) (p4 phl):f2 )
d4
(center (pl ph2) (p3 ph9):f2 )
4u
d6 plO:f2
(center (p2 phi) (p8:spl3 phl):f2)
d6
4u
(pl phi)
DELTA3
(p2 phl)
d13
p16:gp4
d16 pll2:f2 pll6:f3
4u BLKGRAD
go=2 ph31 cpd2:f2 cpd3:f3
dll do:f2 do:f3 mc #0 to 2
FiEA(igrad EA & ip9*2, idO & dd2 &
ip6*2 & ip18*2 & ip31*2)
exit
d13 p2:f2
(pl ph2)
d13
p16:gpl
d16
(p3 ph3):f2
DELTA1
p16 :gp2
d16 plO:f2 plO:f3
(center (pl4:sp3 phll):f2
(p30:sp9 phl2):f3)
DELTA1
p16 :gp2
d16 p2:f2 p3:f3
(p3 phl3):f2
d13
p16:gp5
d16
(p3 phl4):f2
4u
(p21 phl7):f3
DELTA5
(center (p2 phl)
DELTA6
p19 :gp6
; Bloch-Siegert
;ZQ/DQ-Delay
phl=O
ph2=1
ph3=0 0 0 0 2 2 2 2
ph8=
0 2 2
ph9=3 3 1 1
(p22 phl):f3)
d16
4u plO:f2
4u
(center (p14:sp3 phll):f2
phll=O0
ph12=0
183
Appendix
phl3=1
1 1 1 1 1 1 1 3 3 3 3 3 3 3 3
;c13 1. puls
phl4=0
;c13 2. puls;
ph15=l 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
;c13 3. x for ref, y for cross
ph16=1 3
;n15 2. x for ref, y for
cross
ph17=0
;n15 .puls;
phl8=0
;c13 after chem. shift
A1.1.2
evolution ;y for ref, x
for cross
ph31=0
2
2
0
2
0
0
2
2
0
0
2
0
2
2
0
2
0
0
2
0
2
2
0
0
2
2
0
2
0
0
2
r(H2'C2',N)
A1.1.2.1 Reference Experiment
p16 :gpl
dl6
(p3 ph3):f2
d4 plO:f3
d4 plll:fl
(p26 ph26):fl
DELTA1 cpdsl:fl
4u
(center (p4 phl):
;ghcn_quant_c2h2_ref
;avance-version
(00/10/05)
#include <Avance.incl>
#include
Grad.incl>
#include <Delay.incl>
"p2=pl*
2"
"Ilp4=p3*2"
(p30:sp9 phl2):f3)
"p22=p21*2"
4u
DELTA2 p3:f3
"dlO=3u"
"dll=3Om"
"d12=20u"
"d13=4u"
(p3 phl3):f2
d5
4u do:fl
(p26 ph27):fl
(p21 ph14):f3
4u
DELTA5 pll:fl
(CENHN1 p2 phl) (p22 phl):f3
DELTA6
p19:gp6
d16
4u
4u plO:f2
(center (pl4:sp3 phll):f2
(p22 phl2):f3)
4u
"in2O=in0"
"dO=d22"
"d20=d22-d4*2-4u"
"DELTA=dlO*2+p4"
"DELTA1=d22-d4*2-4u-p26"
"DELTA2=d22-4u"
"DELTA3=d13+p16+d16+4u"
"DELTA4=d22-d13 -p16-d16-p2-d4"
"DELTA5=d26/4-4u"
"DELTA6=d26/4-p19-d16-8u"
"d14=d4*2-p26"
"d5=25m/2-p21-d26/2-p26"
4u p2:f2
p19:gp6
d16
DELTA6
(center (p2 phl) (p22 phl):f3)
DELTA5
4u plll:fl
(p2 1 phl6):f3
(p26 ph26):fl
d5 p2:f2 cpdsl:fl
4u
(p3 phl5):f2
dO plO:f3
(center (p4 phl):f
(p30:sp9 phl):f3)
d20
4u do:fl
(p2 6 ph27):fl
d14 pll:fl
(p3 ph8):f2
4u
p16:gp3
"12=tdl/2"
1 ze
dll pll2:f2 pll6:f3
2 dll do:f2 do:f3
3m
3 dll
dll
4 d12 p9:fl
dl cw:fl
4u do:fl
lOu pll:fl
(pl phl)
d4 p2:f2 p3:f3
(center (p2 phl) (p4 phlO):f2)
d4 UNBLKGRAD
(pl ph2)
4u
184
Appendix
d16
phll=0
phl2=0
phl3=l
(pl phi)
d4
lOu
1111111
1111111
0020220
1111111
3 3 3 3
31111111
3 3 3 3 3 3
3333333
33333333
3 3 3 3 3 3
33333333
3 3 3 3 3 3
3333333
0002222
3 3 3 3 3 3 3
1111111
3 3 3 3 3 3 3
1111111
23333333
2
2 2 2 2
3333333
0000000
2222222
1
1 1 3 3
4u
(center (p2 phl) (p4 phl8):f2)
d4
lOu pll2:f2 pll6:f3
4u BLKGRAD
go=2 ph31 cpd2:f2 cpd3:f3
dll do:f2 do:f3 wr #0 if #0 zd
3m ip8
lo to 3 times 2
dll dd0
dll id20
lo to 4 times 12
1
3
3
3
3
ph14=0
ph15=1
1
3
3
ph16=0
2
ph17=0
ph18=0
ph2O=2 2 2
2
2
ph26=1
ph27=3
ph31=0
2 0 0 2 0 2 2 0
exit
phl=0
ph2 = 1
ph3=0 2
ph4 = 0
ph5=0
ph6=0
ph7=1
ph8=0 0 2 2
phlO=0
2202002
A1.1.2.2 Cross Experiment
dll pll2:f2 pll6:f3
2 dll do:f2 do:f3
3m
3 dll
dll
4 d12 p9:fl
dl cw:fl
4u do:fl
lOu pll:fl
(pl phi)
;ghcn_quant_c2h2_cross2
;avance-version
(00/10/05)
#include <Avance.incl>
#include <Grad.incl>
#include <Delay.incl>
"p2=pl* 2 "
I"p4=p3*2"1
"p22=p21* 2 "
d4 p2:f2 p3:f3
(center (p2 phl)
d4 UNBLKGRAD
(pl ph2)
4u
p16 :gpl
dl6
(p3 ph3):f2
"dlO=3u"
"dll=30Om'
"d12=20u"
"d13=4u"
"in2O=in0"
(p4 phlO):f2)
d4 pO:f3
d4 plll:fl
(p26 ph26):fl
DELTA1 cpdsl:fl
4u
(center (p4 phll):
"dO=d22"
"d20=d22-d4*2-4u"
"DELTA=d1O*2+p4"
"DELTA1=d22-d4*2-4u-p26"
"DELTA2=d22 -4u"
(p30:sp9 phl2):f3)
"DELTA3=d13+p16+d16+4u
"DELTA4=d22-d13
-pl6-d6-p2-d4"
4u
DELTA2 p13:f3
(p3 phl3):f2
d5
4u do:fl
(p26 ph27):fl
(p21 phl4):f3
4u
DELTA5 pll:fl
(CENHN1 p2 phl)
"DELTA5=d26/4 -4u"
"DELTA6=d26/4-pi9-d16-8u"
"d14=d4*2-p26"
"d5=25m/2-p21-d26/2-p26"
"dl5=25m/2-p21-d26/2-d4*2-p26"
"12=tdl/2"
1 ze
185
(p22 phl):f3
Appendix
ph14=0 0 0 0 2 2 2 2
ph15= 1 1 1 1 1 1 1
DELTA6
p19 :gp6
d16
4u
4u plO:f2
(center (p14:sp3 phll):f2
(p22 phl2):f3)
4u
1 1 1 1 1 1 1 1
3
3
phl6=1
cross
3
ph17=0
phl8=0
ph20=2
ph26=1
ph27=3
ph31=0
2
4u p2:f2
p19:gp6
d16
DELTA6
(center (p2 phl) (p22 phl):f3)
DELTA5
4u plll:fl
(p21 phl6):f3
d4
; refocussing C2'H2' coupling
d4
(p26 ph26):fl
d15 p2:f2 cpdsl:fl
4u
(p3 phl5):f2
dO plO:f3
(center (p4 phl):f2 (p30:sp9
phl):f3)
d20
4u do:fl
(p26 ph27):fl
d14 pll:fl
(p3 ph8):f2
4u
p16:gp3
d16
(pl phl)
d4
lOu
4u
(center (p2 phl) (p4 phl8):f2)
d4
lOu pll2:f2 pll6:f3
4u BLKGRAD
go=2 ph31 cpd2:f2 cpd3:f3
dll do:f2 do:f3 wr #0 if #0 zd
3m ip8
lo to 3 times 2
dll dd0
dll id20
lo to 4 times 12
exit
phl=0
ph2=l
ph3=0 2
ph4=0
ph5=0
ph6=0
ph7=l
ph8=0 0 2 2
phlO=0
phll=0
phl2=0
phl3=
1 1 1 1 1 11
11111111
11111111
11111111
33333333
33333333
33333333
33333333
3 3 3 3 3 3 3 3
3 3 3 3 3 3 3 3
186
3 3 3 3 3 3 3
3 3 3 3 3 3 3
11 1 11
1 ;1
x for ref, y for
3 3 3 3 3 3 3
2 2 0 2 0 0 2
0 0 2 0 2 2 0
Appendix
A2.1 F-rate calculation
The F-rates were calculated using the CS-tensor values and orientations and the dipole-dipole
vector lengths listed in Table A1.
Nucle-
CS-Tensor
1l
622
633
011
022
033
otfide
Adenine
F
rI'H',NI
- 148ppm
0
Adee n
DD
9
,CSAN9
Guanine
.
DDCSA
CSAC1'N9:4.0
N
-150ppm
______
Cytidine
Ni
.h n ~
C2'H2',NI1/9
pDDCSA
CH N
_________
C1'N9:16.0 °
- 143ppm
C1'N1:17.3°
- 160ppm
C1'N1:31.30
- 148ppm
Adenine
C8H8:67.4°
N
- 150ppm
C8H8:55°
- 143ppm
DAN9
G nne
6/8 H6/8,Ni19
r
Cytidine
Cydne
1
C6H6:78.7
Uracile
Adene
Guann
.9T,
~~ ~iDnC1
,G
Cidi
9H',CI'
C1
ne
63.4ppm*
C1'H1':28.60
~
The
DD, CSA
NH,c
Ga9H6/8,C6/8
Ciidine
Gaie214ppm
GuaCe
Cytidine
0
C6H6:90
11
0
78.8ppm*
C1'H1':116.2 °
106.1ppm*
C1'H1':79.3 °
N9H8:3.8 °
241ppm
C6 NlH6:4.7 °
N9H8:93.8 °
152ppm
N1H6:94.7 °
H6
NH'
Thymine
______
2.15
2.15
215
C
61ppm
N9H8:900
N1H6
2.15
216ppm
C8 C8H8:27°
128ppm
N1H6:95.7 °
136ppm
C8H8:117°
42ppm
NH6:90 °
61ppm
C8H8:90°
C8H8
1.1
8H8:280
241ppm
141ppm
C8H8:118 °
152ppm
59ppm
C8H8:90°
35ppm
C6H6
1.1
238ppm°
N1H6:5.7
0°
238ppm
C6H6:29°
141ppm 59ppm
C6H6:119 0°
128ppm
C6H6:119°
C6H6:90
0
42ppm
C6H6:90°
ipci, dipdebod lghs orthe ddcatl Frates.A ll ndVIhs haw ln
extractdfiUm the programINSIGHT (n-i). The tesor zdues lae wth asteriskshaze ben taken from DFT
aladations[1]. All odxr tensorshazeben obtaidfi0m nridesd
in thesolidstate[2]. In the caseof urad* the
tensorfor 2'dxc.ds
has bn gizen 0 is the angle be n the respetiwe pa r of thedx
CS-tensorand the
iicatad dx &pe zaor. For all tensorsof anutic aton s,1 and S22 ae onernt zdthn the baseplane and S33 S
ppriadar
1.1
N9H8:90 °
35ppm
N1H6:90 °
C6H6:29
Table A1: CS-tensors
and or
C6H6:168.7
C8H8
2.15
2.15
_
Adene
18116/8,C6/8
-29 1ppm
C8H8:90°
-311ppm
11
N9H8
N9H8
Uracile
C
- 199ppm
C8H8:145°
-218ppm
C'HI'
C2'H2'
-28lppm
C8H8:90 °
C1'Hl':81.0°
J~)DCSA
Gaie214ppm
CH,C
C1'N9:90 °
-311ppm
C1'N1:90°
-306ppm
C1'N1:900
C1'H1':111.5
136ppm
N9H8:94.8 °
C8
DDCSA
DDCSA
-291ppm
C1'N9:106.0°
-218ppm
C1'N1:72.7°
-228ppm
C1'N1:58.70
- 198ppm
C8H8:157.4°
C1'H1':23.5
216ppm
N9H8:4.8 °
_denin
/
-28 lppm
C1'N9:900
63.9ppm*
79.3ppm*
108.0ppm*
°
°
_ymme
rl
- 199ppm
Lenth
- 160ppm
-228ppm
-306ppm
C6H6:83.5°
C6H6:173.5 °
C6H6:90 °
110.4ppm*
64.2ppm*
79.9ppm*
C1'H1':21.5 ° C1'H1':107.7 °
C1'H1':78.2 °
'
N9HI'
64ppm*
80.5ppm*
111.2ppm*
0 C1'H1':109.1
°
°
'H1':22.7
C1'H1':78.2
Uracile
I
0
- 198ppm
C1'N9:86.00
Dipole
to it For the C1' tensoronly the pMedion angle orntothe Ci'-Hi bondare k
187
roen
Appendix
All r(HCN)-rates were calculated in an UNIX environment. The coordinates of the three 15N
CS-tensor components as well as the one of the GH vectors were obtained from pdb coordinate
files of the respective nucleotides. The
15
N CS-tensor was simulated by exchanging the N 1 in
pyrimidines and N9 in purines by a phosphorous nucleus, which allowed for the formation of six
bonds. The three additional bonds were used to model the tensor components. For every
nucleotide in both C2ndo and C3-eoo conformation, 72 coordinate files were created by altering
the angle
stepwise by 5 degrees in order to achieve a complete 360° rotation using
<name>.source file in INSIGIffII.
The pdb files were numbered consecutively (format=
<name ><number>.pdb).
From the coordinate files, F(C1'H1',N), F(C2TH2',N) and F(C6H6,N) or F(C8H8,N) were
calculated using the Gprogram get_rate_frompdb.c.
The usage of this program is:
get ratefrom pdb <max_number of pdb_files> <pdbfilename>
The Cprogram was used in combination with a shell script (getrate_frompdb.sh)
in order to
input multiplepdb files.
A1.1.3
Source-file
Source-file for adenine.
# A.source
DefineMacro A
Int n,m
Int zl,z2,z
Get Molecule Archive
Frame 1 /fs/data/ric/GAMMA_HCN/SIMULATION/PDB2/B_Form/A/A.car
A -ReferenceObject
m= -98
Foreach $z2 From 1 To 72
Geometry Dihedral $m A:1:04' A:l:Cl' A:l:P9 A:1:C4
Put Molecule PDB a (a//$z2//.pdb) -Transformed -Displayed -InsightStyle
m = $m + 5
End
End Macro
188
_.
.
Appendix
A1.1.4
Get_rate_frompdb.sh
#!/bin/sh
j =0
until [ $j = 72
do j= echo "$j+l" bc
get-rate_from_pdb 33 a$j.pdb
echo "\r"
done
echo
A1.1.5
\007"
Getratefirmpdb.c
The program calculates the F-rates for purines. In order to obtain the F-rates from pyrimidines, the CB and H 8 have to be
replaced by Q and H.
/* get_ratefromypdb.c
Calculates theta from given pdb file
./
#include "stdio.h"
#include "math.h"
int
int
char
float
float
float
int
char
dum2,dum5;
number;
duml,dum4;
dum9,dumlO;
test_x,test_y,test_z;
testl;
restest,maxno;
atoml[8] ,atom2[8],atom3[8],atom4 [8],atom5 [8] ,atom6[8],atom7[8] ,atom8 [8],atom9
[9];
float
N_x,N_y,N_z,NHl_x,NHl_y,NHlz,NH8_x,NH8_y,NH8_z,Clx,Cly,Cl_z,Hl_x,Hly,Hl_z
float
float
float
float
float
float
float
float
test_neu;
atom3_x,atom3_y,atom3_z,atom4_x,atom4_y,atom4_z;
Sll_x,Sll_y,Sll_z,S22_x,S22_y,S22_z;
C2H2_x,C2H2_y,C2H2_z,betrag_C2H2,H2_x,H2_y,H2_z;
C8H8_x,C8H8_y,C8H8_z,betrag_NH8,H8_x,H8_y,H8_z;
ClHl_x,ClHl_y,ClHl_ z,betrag_NHl;
Nx_x,Nx_y,Nx_z,betrag_Nx,Ny_x,Ny_y,Ny_z,Nz_z,Nz_x,Nz_y,betrag_Ny,betrag_Nz;
sigma_llx,sigmally,sigmall
z,sigma_22 x,sigma_22_y,sigma_22_z,sigma_33_x,
sigma_33_y,sigma 33_z;
float ein _xbetr,ein_y_betr,einzbetr;
float betr sigmall,betr_sigma_22,betr
sigma_33;
float betragl,betrag2,betrag3;
float cos2_THETA,cos2_THETAy;
float
gammac,gamma_c_cl,gammac
c2;
float
s_ll,s_22,s_33,C;
float theta;
float
invcostx, winkel;
char
atomtype[8];
FILE
*fin, *fout;
main (argc,argv)
int argc;
char *argv[];
if (argc
(argc !=
!= 3)
3)
if
189
Appendix
{printf("\n\nget ilratefrom_pdb:
printf("USAGE: get_lratefrompdb
exit(l); }
wrong number of arguments\n\n");
maxNoofAtomsinPDB file.pdb \n\n");
atoml[0]='P';
atoml [1]='19';
atom5 0] = 'H';
atom5[1]='1';
atom5[2] ='\';
atom9[0] = 'H';
atom9[1] =' 8';
sscanf(argv[l] ,"%d",&maxno);
s 11= -150;
s 22= -199;
s_33= -291;
C=
1.6988888;
/*
C=
0.0326999;*/
if ((fin=fopen(argv[2] , "r"))==NULL)
{ printf("ERROR opening file %s !\n",argv[2]); exit(l); }
number=l;
fseek(fin,0,0);
while (number!=maxno) {
fscanf(fin,"%s %d %s %s %d %f %f %f %f %f",&duml,&number,atomtype,
\
&dum4,&restest,&testx,&test_y,&test_z,&dum9,&dumlO);
if (strcmp(&atomtype[0] ,&atoml[0] )==0){
if (restest==l) {
Nx=testx;
N y=test_y;
N_z=testz;
}
}
number= 1;
fseek(fin, 0,0)
while (number<=maxno) {
fscanf(fin,"%s %d %s %s %d %f %f %f %f %f",&duml,&number,atomtype,
\
&dum4,&restest,&testx,&test_y,&test_z,&dum9,&dumlO);
if (strcmp(&atomtype 0] , &atom5[0] ) ==0){
if (restest==l) {
Hl_x=test_x;
Hl_y=test_y;
Hlz=testz;
}
}
number= 1;
fseek(fin,0,0);
while (number<=maxno){
fscanf(fin,"%s %d %s %s %d %f %f %f %f %f",&duml,&number,atomtype,
&dum4,&restest,&test_x, &test y,&test_z,&dum9,&dumlO0);
if (strcmp(&atomtype[0] , &atom9[0] ) ==0) {
if (restest==l) {
H8 x=testx;
H8_y=test_y;
H8_z=testz;
I~~~~
/*
printf("\n\n");
190
\
Appendix
printf("Koordinaten
printf("Koordinaten
printf("Koordinaten
von Atoml(%s): %.3f %.3f %.3f\n", atoml,N_x,N_y,N_z);
von Atoml(%s): %.3f %.3f %.3f\n", atom5,Hl_x,Hl_y,Hl_z);
von Atoml(%s): %.3f %.3f %.3f\n", atom9,H8_x,H8_y,HS_z);
*/
NH8_x = Nx - H8_x;
NH8_y = N y - H8_y;
NH8_z = N_z - H8_z;
betrag_NH8= sqrt(pow(NH8_x,2)+pow(NH8_y,2)+pow(NH8_z,2));
NH1x= Nx
- Hl_x;
NH1_y = N_y - Hl_y;
NHl_z = Nz - Hl_z;
betrag_NHl= sqrt(pow(NH1_x,2)+pow(NHiy,2)+pow(NHlz,2));
cos2_THETA = ((NH1_x * NH8_x) + (NHl_y * NH8_y)
*betrag_NH8);
+ (NHi_z * NH8_z))/(betragNHl
gammac= C * ( (3 * cos2_THETA* cos2_THETA- 1));
winkel= acos (cos2_THETA)*180
printf("%9.3f
/M_PI;
%9.3f %9.3f"',cos2_THETA, gamma_c, winkel)
;
fclose(fin);
Coordinate file independent r-rate calculation
A1.1.6
Cross-correlated relaxation rates were calculated by Gprogramms using the CS-tensor values
and orientations given in Table A1.
A1.1.6.1 r(HC,N)
F(HCN) for adenine in the 14-nt RNA at 600MHz.
/*calculation of HC,N CCRR in Adenine at 600MHz*/
#include
#include
#define
#define
#define
stdio.h>
<math.h>
gammalH
26.7519e7
gammal3C
6.728e7
Hq
1.0546e-34
#define m
/*
gyromag. ratio 1H
[sec* A* kg-1] */
/*
gyromag. ratio 13C [sec* A* kg-1] */
/*
Planck's h/2pi
[J*sec]
*/
0.9999995e-7 /* permeability of vacuum/4/pi [m*kg*sec-2*A-2] */
/* bond angle taken from cns2.0 force field for C3'-endo:*/
char
s1[80];
int
i,j,n,m,l;
double
alpha,beta,beta2,angleO,rch,rco,rop,rcn,
s1Il,s22,s33,
x_h,y_h,zh,
x_ 3 3,y_33,z_33,
x_22,y_22,z_22,
xll,yll,z_11,
x_hp,y_hp,z_hp,
s33_length,s22_length, s11_length,hc_length,
x_dotl,x_dot2,x_dot3,
191
Appendix
costx,costy,costz,invcostx,invcosty,
cosx_out,cosy out,cosz_out,
thetal, theta2, ftheta,
factor,angle_factor,
ni,gammah,tauc,S,omegalSN,
al,a2,c,
rate,rateh8c8,
cosx,cosy,cosz,
x_h8c8,y_h8c8,zh8c8,h8c8_length,
x_dot_h8c8,y_doth8c8,z_dot_h8c8,
costx_h8c8,costyh8c8,costz_h8c8,
fthetah8c8;
main ()
{
alpha = 180-108.7; /*angle between CH-vector and CN bond */
rch=1.09e-10;
rcn=l.5e-10;
omegal5N=2*M_PI*l.Oe6*60.808;
al = -4*(mO*Hq*gammalH*gammal3C*omegal5N)/15;
a2 = rch*rch*rch;
S=1;
/* order paramete r square */
tauc=1.9e-9;
sll=-148;
s22=-198;
s33=-281;
/*all vectors are calculated from N as the origin of the coordinate system
x-axis is along NCl'-bond, z-axis orthogonal to base plane */
/*orientation of H8C8 */
x_h8c8=cos(71.33*M PI/180.0);
y_h8c8=-sin(71.33*M_PI/180);
z_h8c8=0;
h8c8_length=sqrt(x_h8c8*xh8c8+y_h8c8*y_h8c8+z_h8c8*z_h8c8);
/* orientation
of sigmall */
x ll=cos(3.96*M PI/180.0);
y_ll=-sin(3.96*M_PI/180.0);
z_11=0;
sll_length=sqrt(xll*xll+y_ll*yll+zll*z_ll);
/* orientation
of sigma22 */
x_22=cos(93.96*M_PI/180.0);
y_22=-sin(93.96*M_PI/180.0);
z_22=0;
s22_length=sqrt(x_22*x_22+y_22*y_22+z_22*z_22);
/* orientation
of sigma33 */
x_33=0;
y_33=0;
z_33=1;
s33_length=l;
/* calculation
of G(H8C8,N1) */
xdoth8c8=x
h8c8*x ll+yh8c8*y_ll+z_h8c8*z_11;
192
Appendix
y-dot h8c8=x_h8c8*x_22+y_h8c8*y_22+z_h8c8*z22;
z_dot_h8c8=x_h8c8*x_33+y_h8c8*y_33+z_h8c8*z_33;
costx_h8c8=(x_dot_h8c8/(h8c8_length*sll_length));
costy_h8c8=(y_doth8c8/(h8c8_length*s22
length));
costz_h8c8=(z_dot_h8c8/(h8c8_length*s33_length));
fthetah8c8=0.5*(sll*le-6*(3*costxh8c8*costx h8c8-l)+s22*le6*(3*costy_h8c8*costyh8c8-1)+s33*1e-6*(3*costz_h8c8*costz_h8c8-1));
rate_h8c8=(al*ftheta_h8c8*tauc)/a2;
/* calculation
of G(Hl'Cl',N9)
*/
for(j=0;j<360;j++){
angleo=(double)(j);
/*definition
of chi*/
beta2=angleO-240;
/*orientation
of the Hl'Cl'-vector*/
xh=rch*cos(alpha*MPI/180.0); /* x-position is fixed */
yh=rch*sin(alpha*MPI/180.0)*cos(beta2*M_PI/180.0);
z_h=rch*sin(alpha*MPI/180.0)*sin(beta2*M_PI/180.0);
hclength=sqrt((x_h*xh)+(y_h*y_h)+(z_h*z_h));
/*angle between
sigmacomponents
and Cl'Hl'
*/
xdotl=xh*xll+y_h*y_ll+z_h*z_11;
x_dot2=x_h*x_22+y_h*y_22+zh*z_22;
x_dot3=x_h*x_33+y_h*y_33+z_h*z_33;
/* projection angle */
costx=(x_dotl/(hc
length*sll_length));
costy=(xdot2/(hc length*s22_length));
costz=(x_dot3/(hclength*s33_length));
/* relaxation rate */
ftheta=0.5*(sll*1.Oe-6*(3*costx*costx-l)+s22*1.0e-6*(3*costy*costy-l)+s33*1.0Oe6*(3*costz*costz-1));
printf("%d
%e %e \n",j,rate,
rate_h8c8);
A1.1.6.2 (HN,C) for the base moiety
/*calculation of the unwanted HN,C CCRR in Adenine at 600MHz*/
#include <stdio.h>
#include <math.h>
#define gammalH
26.7519e7
/*
gyromag. ratio H
[sec* A* kg-l */
#define gammal5N
-2.75e7
/*
gyromag. ratio 15N [sec* A* kg-l */
#define gammal3C
6.728e7
/*
gyromag. ratio 13C [sec* A* kg-l */
#define Hq
1.0546e-34
/*
Planck's h/2pi
[J*sec]
*/
#define m
0.9999995e-7 /*permeability of vacuum/4/pi [m*kg*sec-2*A-2]*/
/* bond angle taken from cns2.0 force field for C3'-endo:*/
193
Appendix
char
int
double
sl ([80];
i,j,n,m,1;
alpha,rch,
al, a2,
sll,s22,s33,
thetall,theta22,
ftheta,
theta33,
ni,gammah,tauc,S,omegal5N,
ratehnc;
main ()
{
rch=2.15e-10;
omegal5N=2*MPI*1.0e6*70.947;
al -4*(mO*Hq*gammalH*gammal3C*omegal5N)/15;
a2 = rch*rch*rch;
/* order parameter
S=1;
tauc=1.9e-9;
/*----------------------------------
ADENINE
square */
--------------------------------------
- ,/
s11=216;
s22=136;
s33=61;
thetall=cos(4.8*M_PI/180.0);
theta22=cos(94.8*M_PI/180.0);
theta33=cos(90*M_PI/180.0);
ftheta=0.5*(sll*le-6*(3*thetall*thetall-1)
1)+s33*1e-6*(3*theta33*theta33-1));
+s22*1e-6*(3*theta22*theta22-
ratehnc=(al*ftheta*tauc)/a2;
printf("G(HNC) for adenine= ");
printf(" %e \n",rate_hnc);
%e %e \n", sl, s22, s33);
printf("%e
printf("%e
%e %e \n", 3*thetall*thetall-1,
3*theta33*theta33-1);
printf(" %e \n",ftheta);
printf(" %e %e \n",al, a2);
/*----------------------------------
3*theta22*theta22-1,
GUANINE --------------------------------------
sll=214;
s22=141;
s33=59;
thetall=cos(3.8*M_PI/180.0);
theta22=cos(93.8*M_PI/180.0);
theta33=cos(90*M_PI/180.0);
ftheta=0.5*(sll*le-6*(3*thetall*thetall-1)
1)+s33*1e-6*(3*theta33*theta33-1));
+s22*1e-6*(3*theta22*theta22-
ratehnc=(al*ftheta*tauc)/a2;
printf("G(HCN) for guanine= ")
printf(" %e \n",ratehnc);
%e %e \n", 3*thetall*thetall-1,
printf("%e
3*theta33*theta33-1);
printf(" %e \n",ftheta);
194
-
3*theta22*theta22-1,
Appendix
/*----------------------------------
CYTOSINE -------------------------------------
*/
--
s11=241;
s22=152;
s33=35;
thetall=cos(4.7*M_PI/180.0);
theta22=cos(94.7*MPI/180.0);
theta33=cos(90*M_PI/180.0);
ftheta=0.5*(sll*le-6*(3*thetall*thetall-1)+s22*1e-6*(3*theta22*theta221)+s33*1e-6*(3*theta33*theta33-1));
ratehnc=(al*ftheta*tauc)/a2;
printf("G(HCN) for cytosine= ");
printf(" %e \n",rate_hnc);
printf("%%e
e %e \n", 3*thetall*thetall-1,
3*theta33*theta33-1);
printf(" %e \n",ftheta);
/*----------------------------------
3*theta22*theta22-1,
THYMINE --------------------------------------
s11=238;
s22=128;
s33=42;
thetall=cos(5.7*M_PI/180.0);
theta22=cos(95.7*M_PI/180.0);
theta33=cos(90*M_PI/180.0);
ftheta=0.5*(sll*le-6*(3*thetall*thetall-l)+s22*1e-6*(3*theta22*theta221)+s33*1e-6*(3*theta33*theta33-1));
ratehnc=(al*ftheta*tauc)/a2;
printf('"G(HCN) for thymine=
);
printf(" %e \n",ratehnc);
printf("%%e
e %e \n", 3*thetall*thetall-1,
3*theta33*theta33-1);
printf(" %e \n",ftheta);
}
3*theta22*theta22-1,
/*calculation of the unwanted HN,C CCRR in Adenine at 600MHz*/
#include <stdio.h>
#include <math.h>
#define gammalH
26.7519e7
/*
gyromag. ratio 1H
[sec* A* kg-1] */
#define gammal5N
-2.75e7
/*
gyromag. ratio 15N [sec* A* kg-1] */
#define gammal3C
6.728e7
/*
gyromag. ratio 13C [sec* A* kg-1] */
#define Hq
1.0546e-34
/*
Planck's h/2pi
[J*sec]
*/
#define m
0.9999995e-7
/* permeability of vacuum/4/pi
m*kg*sec-2*A-2] */
A1.2 References
1.
Fiala, R; Czernek J.;Sldenar, V. (2000). Transverse relaxation optimized triple-resonance
2.
NMR experiments for nucleic acids.JBiwr NMR, 16,291-302.
Stueber, D. and Grant, D. M. (2002). (13)C and (15)N chemical shift tensors in
adenosine, guanosine dihydrate, 2'-deoxythymidine, and cytidine.JAmbemnSaoc, 124,
10539-10551.
195
Appendix
CHAPTER 3: RNA Tetraloop Dynamics
A2
A2.1 Pulse sequences
A2.1.1
R1
;hsqctletf2gpsi
;avance-version (03/01/27)
;2D H-1/X correlation via double inept transfer
using sensitivity improvement
;for measuring C-13 T1 relaxation times
;phase sensitive using Echo/Antiecho-TPPI gradient selection
;with decoupling during acquisition
;using f3 - channel
;using flip-back pulse
;(use parameterset HSQCT1ETF3GPSI)
prosol relations=<triple>
d26 UNBLKGRAD
(pl ph2)
#include <Avance.incl>
#include <Grad.incl>
#include <Delay.incl>
"p2=pl*2"
4u plO:fl
(pll:spl phl:r):fl
4u
p16:gpl
d16 pll:fl
"p4=p3*2"
(p3 ph3):f2
d25
(center (p2 phl) (p4 phl):f2
d25 pll9:fl
(p3 ph2):f2
"dO=3u"
"dll=30m"
"d24=ls/(cnst4*cnstll)"
(p26 ph2):fl
TAU cpdsl:fl phl
4u do:fl
(p26 ph7):fl
4u
p16:gp4
d16 pll:fl
"d25=ls/(cnst4*cnstl2)"
"d26=ls/(cnst4*4)"
"DELTA2=p19+d16+8u"
"DELTA3=d25-p19-d16-8u-p8"
#
(p3 ph8):f2
4u
ifdefLABELCN
p19:gp2*-l*EA
"DELTAl=d25-p19-d16-larger(p2,p8)d0*2"
#
else
"DELTAl=d25-p19-d16-p2-dO*2"
#
endif /*LABEL_CN*/
d16 plO:f2
(pB:spl3 phl):f2
4u
DELTA3 p2:f2
(p4 ph6):f2
dO
(center (p2 ph9) (p8:spl3 phl):f2
#
else
(p2 ph9)
#
endif /*LABEL_CN*/
1 ze
dll pll2:f2 stO
2 6m do:f2
3 3m
4 dll
5 dll
"d7=vd"
"TAU=d7-p26*2-p16-d16-8u"
dl
(pl phl)
dO
p19:gp2*EA
d16
DELTA1
(center (pl phl)
d24
(center (p2 phl)
d26 p2:f2
(center (p2 phl)
ifdefLABELCN
#
aqseq 312
(p4 phl) :f2 )
196
(p3 ph4):f2
)
(p4 phl):f2
)
Appendix
d24
(center (pl ph2) (p3 ph5):f2
d26
(center (p2 phl) (p4 phl):f2
d26
(pl phi)
DELTA2
(p2 phi)
4u
pl9:gp3
d16 pll2:f2
4u BLKGRAD
)
dll mc #0 to 5
F2EA(igrad EA & ip5*2, idO &
ip6*2 & ip8*2 & ip3l*2)
)
exit
phO=0
phl=0
ph2=1
ph3=0 0 0 0 2 2 2 2
ph4=0 0 2 2
ph5=3 3 1 1
ph6=0
ph7=3
ph8=l 3
ph9=0 0 2 2
ph31=0 2 2 0 2 0 0 2
goscnp ph31 cpd2:f2
3m do:f2
3m st ivd
lo to 3 times nbl
3m ipp3 ipp4 ipp5 ipp8 ipp9 ipp31
lo to 4 times ns
;d7 : delay for inversion recovery
[30 msec]
;dll: delay for disk I/O
;d16: delay for homospoil/gradient recovery
;d24: 1/(4J)YH for YH
; 1/(BJ)YH for all multiplicities
;d25: 1/(4J)YH for YH
; 1/(8J)YH for all multiplicities
;d26: 1/(4J(YH))
;cnst4: = J(YH)
;cnstll: for multiplicity selection = 4 for NH, 8 for all multiplicities
;cnstl2: for multiplicity selection = 4 for NH, 8 for all multiplicities
;in0: 1/(2 * SW(X)) = DW(X)
;ndO: 2
;NS: 1 * n
;DS: >= 16
;tdl: number of experiments
;FnMODE: echo-antiecho
;cpdsl: decoupling according to sequence defined by cpdprgl
;cpd3: decoupling according to sequence defined by cpdprg3
;pcpdl: fl channel - 90 degree pulse for decoupling sequence
;pcpd3: f2 channel - 90 degree pulse for decoupling sequence
;use gradient
ratio:
gp 1 : gp 2 : gp 3 : gp 4
50 :
80 : 16.2 :
30 for N-15
;for z-only gradients:
;gpzl: 50%
;gpz2: 80%
;gpz3: 16.2%
;gpz4: 30%
;preprocessor-flags-start
;LABEL_CN: for C-13 and N-15 labeled samples start experiment with
;
~ option -DLABEL CN (eda: ZGOPTNS)
;preprocessor-flags-end
;$Id: hsqctletf2gpsi,v
1.1.2.2 2003/01/31 11:45:02 ber Exp $
197
Appendix
A2.1.2
Rlp
;hsqctretf2gpsi.t5
;avance-version (03/04/25)
;2D H-1/X correlation via double inept transfer
using sensitivity improvement
;for measuring C-13 Trho relaxation times
;phase sensitive using Echo/Antiecho-TPPI gradient selection
;with decoupling during acquisition ;using f3 - channel ;using flip-back pulse
;(use parameterset HSQCTRETF3GPSI)
;K.T. Dayie & G. Wagner, J. Magn. Reson. A 111, 121-126 (1994)
;F.A.A. Mulder, R.A. de Graaf, R. Kaptein & R. Boelens,
; J. Magn. Reson. 131, 351-357 (1998)
. Millet, D.A. Torchia &
;D.M. Korzhnev, N.R. Skrynnikov,
; L.E.Kay, J. Am. Chem. Soc. 124, 10743 (2002)
(pl phl)
d26 p12:f2
(center (p2 phl) (p4 phl):f2
d26 UNBLKGRAD
(pl ph2)
4u
p16:gpl
d16 pll:fl
(p3 ph3):f2
d25
(center (p2 phl) (p4 phl):f2
d25 pll9:fl
(p3 phll):f2
;phaseOnTcu
prosol relations=<triple>
#include <Avance.incl>
#include <Grad.incl>
#include <Delay.incl>
"Ip2=pl*2"
"p4=p3*2"
"p22=p2 l* 2 "
"dO=3u"
"dll=30m"
"dl2=20u"
"d24=ls/(cnst4*cnstll)"
"d25=ls/(cnst4*cnstl2)"
"d26=ls/(cnst4*4)"
p16 :gp5
d16 plO:f2
4u fq=cnst30(sfo hz):f2
(p 2 9:spl9 phl):f2
4u
4u p125:f2
4u cpds8:f2 ph8
"cnst29=0"
"DELTA2=p19+dl6+8u"
"DELTA3=d25-p19-d16 -p8 - 8u"
"DELTA4=d24-p19-d16"
"DELTA5=d26-pl6-dl6"
#
"p30=random(p31,cnst31)"
6 (p30 phlOA) :fl
"d31=d31-p30"
"p30=random(p31,cnst31)"
if "d31 < p30"
{
"p30=d31"
if "p30 < 3u"
{
"p30=3u"
}
goto 7
}
else
{
goto 6
ifdef LABEL CN
"DELTAl=d25-pl9-dl6-larger(p2,p8)
d0*2-4u"
else
#
"IDELTAl=d25-pl9-dl6-p2-dO*2
#
endif /*LABEL_CN*/
aqseq 312
1 ze
dll p112:f2 stO
2 6m do:f2
3 3m
4 dll
5 dll p125:f2
7 (p30 phlO
^)
:fl
"d31=vd"
dl fq=cnst29(sfo hz):f2
4u pll:fl
4u do:f2
4u plO:f2
(p29:sp20 phl):f2
4u
4u fq=cnst29(sfo hz):f2
30m
p16 :gp4
198
)
)
Appendix
dl6
(pl phi)
DELTA2
(p2 phi)
4u
p19 :gp3
d16 pll2:f2
4u BLKGRAD
goscnp ph31 cpd2:f2
d16 p12:f2
(p3 phl2)::f2
4u
4u p10:f2
(p8:spl3 phl):f2
p19 :gp2*-]*EA
d16
DELTA3 pll:fl p2:f2
(p4 ph6):f2
dO
4u plO:f2 p3:f3
#
ifdefLABELCN
(center (p2 ph7)
#
3m do:f2
3m st ivd
lo to 3 times nbl
3m ipp3 ipp4 ipp5 ipp6 ipp7 ipp31
lo to 4 times ns
(p8:spl3 phl):f2
(p22 phl):f3)
else
dll mc #0 to 5
F1QF()
F2EA(igrad EA & ip5*2 , id0 & ipl2*2 &
ip31*2)
exit
(p2 ph7)
#
endif /*LABEL_CN*/
dO
p19 :gp2*EA
d16 p2:f2
DELTA1
(center
p19 :gp6
d16
DELTA4
(center
DELTA4
p19:gp6
d16
(center
p16:gp7
d16
DELTA5
(center
DELTA5
p16:gp7
(pl phl) (p3 ph4):f2
)
phO=0
phl=0
ph2=1
ph3=0 2
(p2 phl) (p4 phl):f2
)
ph4=0
ph5=3
ph6=0
ph7=0
(pl ph2)
(p3 ph5):f2
)
(p:2phl)
(p4 phl):f2
)
ph8=0
ph9=2
phlO=l 3
phll=l
phl2=1
ph31=0
0
3
0
0
0
3
1
0
0
3
1
0
2
1
2
2
2
1
2
2
2
1
3
2
2
1
3
2
2 2 0 2 0 0 2
;d20: wanted Trho delay
;d21: maximum Trho delay in series
;d24: 1/(4J)YH for YH
; 1/(8J)YH for all multiplicities
;d25: 1/(4J)YH for YH
; 1/(8J)YH for all multiplicities
;d26: 1/(4J(YH))
;cnst4: = J(YH)
;cnstll: for multiplicity selection = 4 for NH, 8 for all mu:Itiplicities
;cnstl2: for multiplicity selection = 4 for NH, 8 for all mu.ltiplicities
;cnst28: on resonance frequency f2 [in ppm]
f2 [in ppm]
;cnst29: off resonance frequency
;cnst30: difference in frequency [in Hz]
;14: loop for Trho delay
;15: loop for compensation of heating effects = 14(max) - 14 (current)
;in0: 1/(2 * SW(X)) = DW(X)
;nd0: 2
;NS: 1 * n
;DS: >= 16
;tdl: number of experiments
;FnMODE: echo-antiecho
;cpd3: decoupling according to sequence defined by cpdprg3
;pcpd3: f2 channel - 90 degree pulse for decoupling sequence
;use gradient ratio:
gp
30 :
1 : gp
2 : gp 3
80 : 16.2
for N-15
;for z-only gradients:
199
Appendix
;gpzl:
;gpz2:
;gpz3:
;gpz4:
;gpz5:
;gpz6:
;gpz7:
30%
80%
16.2%
50%
-40%
5%
-2%
;use gradient files:
;gpnaml: SINE.100
;gpnam2: SINE.100
;gpnam3: SINE.100
;gpnam4: SINE.100
;preprocessor-flags-start
;LABELCN: for C-13 and N-15 labeled samples start experiment with
;
~ option -DLABEL_CN (eda: ZGOPTNS)
;preprocessor-flags-end
;$Id: hsqctretf2gpsi,v
1.1.2.1 2002/07/16 12:59:36 ber Exp $
200
Appendix
A2.1.3
3C HetnOe
;invinoef2gpsi
;avance-version
;2D H-1/X correlation via double inept transfer
;using sensitivity improvement
;for measuring Hl-C13 NOEs
;phase sensitive using Echo/Antiecho-TPPI gradient selection
;recording NOE and NONOE interleaved
#include <Avance. incl>
#include <Grad.incl>
#include <Delay.incl>
p16 :gp2*-l*EA
dl6
DELTA3
(p4 ph6):f2
4u
dO plO:f2 p3:f3
(CEN_HC2 p2 ph9):fl
22
phl):f3
(CENCN1 p
(p14:sp3 phl):f2
dO
4u p12:f2
"pO=pl* 4 /3"
"p2=pl*2"
"p4=p3*2"
"dO=3u"
"dll=30m"
"d12=20u"
"d24=ls/(cnst4*cnstll)"
"d25=ls/(cnst4*cnstl2)"
"d26=ls/(cnst4*4)"
p16:gp2*EA
d16
DELTA1
(CEN_HN1pl phl) (p3 ph4):f2
d24
4
(CENHN2 p2 phl) (p phl):f2
d24
(CEN_HN1 pl ph2) (p3 ph5):f2
d26
(CENHN2 p2 phl) (p4 phl) :f2
d26
(pl phl)
DELTA2
(p2 phl)
4u
p16:gp3
d16 pll2:f2
4u BLKGRAD
go=2 ph31 cpd2:f2
dll do:f2 wr #0 if #0 zd
"DELTAl=d25-p16-d16-p14-dO*2-4u"
"DELTA2=p6+dl6+8u"
"DELTA3=d25-p16-d16- 8u-p14"
"CENHC2=(p14-p2)/2"
"CEN HNi=(p3-pl)/2"
"CEN HN2=(p4-p2)/2"
"CENCNl=(p14-p22)/2"
"10=0"
"13= (tdl/4)
"14=dl/(pO*4/3+5m)
1 ze
dll pl2:f2
2 dll do:f2
6m
3 3m
4 dll
12m
5 d12
6m ip5*2 igrad EA
lo to 3 times 2
3m iu0
lo to 4 times 2
if "10 %2 .= 1" goto 100
dll idO ip6*2
6m ip8*2
6m ip31*2
lo to 5 times 13
6 (p0 phl)
5m
lo to 6 times 14
4u
goto 101
exit
phl=0
ph2=1
ph4=0 0 2 2
ph5=3 3 1 1
ph6=0
ph7=3
ph8=1 3
ph9=0 0 2 2
ph31=0 2 2 0
100 dl
101 50u UNBLKGRAD
(pl phi)
4u
p16:gpl
d16 pl2:f2
(p3 ph8):f2
4u
(pl4:sp3 phl):f2
201
Appendix
A2.2 Extraction of peak intensities
The spectra were transferred to felix (msi) and the peak intensities were extracted from the
picked peaks by a grid search using the macro autopkhpos.mac
A2.2.1
Autopkhpos.mac
c** autopkhpos.mac - Oz TSRI Jan 1997
c** get peakheights for only positive peaks
c** use for analysis of T2 and Tlrho data
def fitm 1
; first item
def litm 14
; last peak numer
def smxfil tlr
; dataname
for count 1 10
; 1st and last number of datasets
mat &smxfil&count.mat
ty opening &smxfil&count.mat
for ii &fitm &litm
dba element load xpk:peaks.&ii.cenl poil
dba element load xpk:peaks.&ii.cen2 poi2
def wdthl 4
;gridsearch 6 points
def wdth2 4
;gridsearch 6 point
def pkht 0.0
def temp 0.0
def maxp 0.0
for nrow 0 &wdth2
eva row (&poi2+&nrow-(&wdth2)/2)
loa 0 &row
for ncol 0 &wdthl
eva col (&poil+&ncol-(&wdthl)/2)
gv &col temp
if &temp t &maxp againa
eva maxp &temp
againa:
next
next
ty &ii &maxp $
dba element store vol:volumes.&ii.vol&count
&maxp
esc out
if &out ne 0 escape
next
cmx
next
escape:
ex &return
end
202
Appendix
A3
CHAPTER 4: Structure of the TCR 4-chain
A3.1 Assignment of free TGR 4 at 278K
298K
278K
Residue
N
HN
Ca
40.45
55.30
52.36
53.14
59.19
53.45
55.00
54.87
53.24
55.74
50.03
51.38
48.07
60.38
49.83
55.31
52.71
53.45
42.69
53.24
50.86
53.66
53.14
55.31
50.55
54.17
54.07
50.55
52.88
42.48
53.35
53.45
53.76
54.48
55.00
51.48
60.59
52.80
52.00
53.97
54.07
53.66
42.59
1
2
3
4
5
6
7
8
9
10
11
12
13
115.17
124.09
122.75
121.92
125.11
121.71
117.99
122.90
117.47
125.27
118.00
124.59
8.50
8.27
8.23
7.99
8.17
8.15
8.05
8.25
8.24
8.24
7.93
7.80
123.34
118.74
122.15
121.33
109.92
119.12
118.89
120.15
121.79
119.62
119.90
120.81
121.13
117.84
121.41
108.27
8.19
7.89
7.94
8.16
8.32
8.03
8.32
8.14
7.96
7.91
8.00
8.20
7.93
8.05
7.97
8.17
7.86
8.27
8.39
8.18
7.87
8.20
7.82
8.00
7.87
8.16
8.10
7.87
8.17
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
120.07
122.60
121.57
120.80
119.63
121.71
119.84
122.37
120.15
122.38
119.56
120.13
109.23
203
CB
C,
60.94
39.55
27.89
30.15
30.27
36.94
60.83
28.11
60.83
16.12
38.30
15.44
29.47
16.23
35.93
26.98
26.42
166.85
178.01
172.70
174.87
175.38
175.16
176.02
178.96
174.32
177.65
172.31
175.74
26.76
36.04
26.53
39.44
35.93
35.81
27.21
36.04
39.32
28.00
27.89
27.44
27.28
35.93
38.55
29.59
38.87
38.42
29.47
27.32
27.66
cI
173.69
171.73
175.41
176.05
173.75
178.10
175.02
176.50
175.02
172.50
175.48
176.38
174.89
172.39
173.59
171.01
178.79
174.35
174.28
174.20
174.52
175.83
173.78
173.78
172.20
173.14
172.45
173.44
173.31
Appendix
44
45
119.63
122.87
7.92
8.26
52.93
49.93
28.34
37.96
179.26
175.08
46
--
--
60.83
29.36
--
47
48
49
50
51
119.59
120.53
109.72
108.04
121.26
8.34
8.10
8.29
8.07
7.92
53.86
52.93
42.59
42.38
51.69
26.98
35.81
--29.47
172.75
173.14
173.03
177.49
179.32
52
--
--
59.93
--
--
53
54
55
56
121.79
122.83
123.34
121.11
8.35
8.28
8.34
8.45
53.14
53.04
53.40
48.28
28.11
28.11
30.49
35.81
173.52
174.47
174.85
174.91
57
--
--
60.94
29.25
--
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
118.14
121.27
109.60
120.81
119.86
120.00
120.87
121.34
119.93
121.72
120.14
121.05
120.00
123.85
119.40
123.71
118.82
116.95
122.45
120.44
111.84
119.27
121.35
109.20
120.46
121.94
121.94
122.00
109.76
120.23
109.54
118.29
120.31
108.42
120.81
120.51
122.37
108.56
120.75
8.16
7.93
8.23
7.74
8.02
8.06
8.18
7.97
7.99
8.02
8.14
7.97
8.08
7.92
8.07
7.98
7.92
7.79
8.18
7.86
8.26
7.98
8.24
8.17
8.13
8.25
8.21
8.26
8.29
8.15
8.35
8.23
8.21
8.18
7.81
8.10
8.10
7.60
7.94
53.35
53.86
42.69
52.31
55.31
50.46
26.08
26.98
-39.59
36.04
36.04
172.97
174.88
173.18
178.83
172.57
175.63
176.51
176.33
171.52
174.28
173.43
173.75
172.53
174.08
170.73
173.89
171.67
175.05
178.48
173.58
172.89
177.96
173.64
172.61
178.60
173.81
174.26
174.53
173.47
179.13
172.48
178.64
177.49
171.01
178.66
172.48
175.22
175.16
179.27
.
52.80
53.24
54.18
51.59
53.97
53.24
50.29
54.17
50.03
55.41
55.52
53.86
58.72
42.59
53.06
53.55
42.59
53.66
53.49
53.40
53.45
42.59
53.66
42.48
53.32
51.76
42.59
52.41
55.31
52.93
42.28
52.41
204
39.14
26.30
30.15
38.19
29.81
29.59
16.23
27.32
16.23
35.81
60.94
27.32
35.70
-29.93
29.81
-27.32
27.90
27.78
27.89
-30.15
-26.76
38.30
-39.55
29.81
26.76
-39.89
Appendix
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
116.50
115.47
125.63
113.63
123.12
120.59
113.78
122.23
121.87
123.27
117.25
116.96
119.92
120.68
125.05
122.82
8.28
8.01
8.05
7.96
8.17
8.17
7.77
8.03
8.16
7.95
7.72
7.70
7.94
8.13
8.09
8.06
55.52
59.14
50.03
59.35
54.07
52.10
59.66
56.35
54.90
51.24
52.93
53.14
53.06
52.97
49.52
50.04
60.94
66.49
16.49
66.80
30.15
37.96
66.72
35.81
37.96
37.85
39.21
25.96
29.81
26.53
16.45
38.87
171.59
176.88
178.10
170.93
177.24
174.22
173.67
177.82
174.64
173.81
169.79
171.78
177.84
174.49
176.13
172.38
7.80
60.15
54.53
28.91
174.75
113
114
115
125.86
Table A2:Assigvrm ff
A3.2 Assignment of free TCR
4
TCR at 278K
at 317K
310K
Residue
N
HN
124.03
122.18
120.71
124.26
121.14
8.54
8.52
8.16
8.41
8.35
122.87
8.55
125.19
118.19
124.29
8.50
8.29
8.09
50.53
51.90
48.37
123.06
117.97
121.46
120.96
109.59
119.30
118.87
120.06
121.87
119.30
119.64
120.70
120.97
8.43
8.09
8.27
8.45
8.58
8.39
8.61
8.46
8.28
8.18
8.36
8.54
8.26
50.44
55.65
53.45
54.19
43.16
53.80
51.34
54.01
53.24
55.87
51.21
54.83
53.37
CB
C,
4.26
3.99
4.13
169.36
172.13
171.05
170.72
170.93
1
2
3
4
5
6
7
53.11
53.71
59.78
54.06
55.22
8
9
171.24
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
4.46
169.50
172.28
170.53
4.26
-
205
4.57
4.19
4.16
4.21
4.55
4.17
4.15
5.06
4.54
4.08
4.17
171.81
172.65
170.83
170.64
171.55
169.33
170.94
170.85
172.16
170.71
170.27
171.72
Appendix
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
118.10
121.41
108.26
120.07
122.43
121.55
120.63
119.48
121.44
119.01
122.87
120.02
121.48
119.63
120.25
109.07
119.70
122.60
8.43
8.27
8.50
8.20
8.56
8.73
8.52
8.17
8.49
8.09
8.33
8.27
8.38
8.42
8.26
8.47
8.26
8.50
51.13
-43.12
53.67
54.06
54.62
54.53
55.61
52.20
60.69
53.50
52.33
54.75
54.49
54.27
43.08
53.58
50.48
4.19
3.83
4.27
4.18
4.14
4.11
-4.52
3.94
4.16
4.43
4.12
4.15
4.19
3.85
---
172.27
170.25
172.92
169.05
171.26
171.34
171.44
171.22
170.59
171.50
171.51
172.44
171.86
172.19
171.72
171.82
168.85
170.81
--
46
--
--
--
4.27
172.15
47
48
49
50
51
121.07
120.23
109.35
108.06
121.22
8.58
8.41
8.58
8.39
8.21
54.53
53.58
43.33
42.86
51.90
-4.37
3.87
3.85
--
172.24
171.95
171.91
169.71
168.83
52
--
--
--
4.30
--
53
54
55
56
121.44
122.18
122.85
120.87
8.60
8.50
8.58
8.70
53.76
4.21
54.06
48.98
4.21
-4.18
--
171.84
171.24
171.00
170.95
57
--
--
--
4.27
--
58
59
60
61
62
63
118.01
120.96
109.32
120.95
119.48
119.82
8.48
8.26
8.52
8.07
8.28
8.39
53.93
54.87
43.20
53.15
55.69
51.21
4.21
4.15
3.82
4.17
4.14
--
172.08
171.08
171.95
169.04
172.14
170.72
64
--
--
--
--
--
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
121.29
119.79
121.56
119.97
120.74
119.85
123.71
119.06
123.45
118.38
116.49
122.32
119.96
111.33
119.44
121.22
8.28
8.32
8.32
8.48
8.29
8.41
8.26
8.39
8.26
8.19
8.16
8.52
8.16
8.54
8.31
8.51
53.32
53.71
54.87
52.46
54.44
53.54
50.78
54.66
50.35
55.95
56.04
54.66
59.53
43.12
53.45
54.40
4.18
4.18
4.12
4.48
4.16
4.30
171.36
4.09
--4.24
4.20
-3.84
4.34
4.21
171.66
172.59
171.29
171.62
171.57
172.08
-173.11
171.53
172.62
171.00
169.42
171.74
171.99
169.47
171.51
206
Appendix
81
82
83
84
85
86
87
88
109.06
120.57
121.71
121.60
121.78
109.59
120.38
109.47
-4.17
--
43.08
54.44
54.01
53.88
54.01
43.12
54.10
42.95
8.48
8.48
8.55
8.49
8.52
8.59
8.43
8.63
--
-3.87
4.21
--
172.12
169.17
171.62
171.32
171.17
171.67
168.87
172.14
89
--
--
--
--
--
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
120.72
108.49
121.02
120.03
121.84
108.71
120.97
116.20
114.96
125.57
112.90
122.72
120.56
113.23
121.90
121.63
123.44
118.05
117.35
119.72
120.21
124.55
122.45
8.53
8.51
8.14
8.33
8.39
8.09
8.21
8.54
8.26
8.38
8.22
8.45
8.48
8.10
8.33
8.39
8.27
8.15
8.15
8.24
8.41
8.34
8.25
52.33
43.38
53.11
55.65
53.67
43.12
52.94
56.12
59.61
50.40
59.57
54.36
52.46
59.83
56.34
52.16
51.21
4.10
4.49
3.81
4.18
4.37
4.18
3.80
4.31
53.63
-49.92
50.61
-4.20
4.00
--
171.71
169.53
169.19
172.19
170.85
171.07
168.92
172.62
169.95
169.35
172.87
169.70
171.26
169.47
169.49
170.81
171.39
173.39
172.70
170.02
171.14
170.33
172.11
113
114
---
---
---
-4.31
---
115
125.53
8.01
55.09
--
171.09
--
4.32
4.37
4.30
-4.52
4.21
-4.41
---
--
Table A3: A ssigmrntqffie TCR (at310K
A3.3 Assignment of TCR 4/LMPG at 3 10K
~/LMPG
310K
Residue
N
HN
41.33
56.07
53.21
54.12
59.77
53.71
55.22
55.75
53.79
56.31
50.69
1
2
115.04
123.57
3
120.51
4
118.44
5
122.76
6
119.88
7
115.57
8
9
122.26
116.30
10
11
125.27
~ ~~~~~~~~~~~~~~~~~.,
8.58
8.42
8.20
7.74
7.91
8.17
8.01
8.44
8.32
8.32
207
CB
C
61.97
40.34
28.53
30.68
31.17
37.69
62.05
28.61
61.72
16.97
115.04
123.57
120.51
118.44
122.76
119.88
115.57
122.26
116.30
125.27
I
Appendix
12
13
117.28
123.91
8.10
7.86
51.67
48.71
38.85
15.98
117.28
123.91
-
--
--
--
15
16
17
18
19
20
21
22
23
122.40
117.20
120.69
119.97
108.88
119.52
118.18
119.99
120.69
8.33
7.87
8.04
8.25
8.39
8.28
8.34
8.34
8.18
50.95
56.24
54.40
54.50
43.69
54.79
52.21
55.10
55.02
16.81
36.13
26.96
26.80
-26.88
36.46
26.63
39.76
122.40
117.20
120.69
119.97
108.88
119.52
118.18
119.99
120.69
24
25
---
---
---
---
---
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
118.98
118.35
116.92
120.14
107.26
119.52
121.18
120.44
119.41
118.79
121.09
118.62
121.05
119.42
120.60
119.34
119.88
108.83
119.59
122.69
8.20
7.85
8.00
8.06
8.32
8.00
8.22
8.54
8.35
7.92
8.24
7.92
8.04
8.05
8.14
8.19
8.09
8.31
8.06
8.35
55.12
53.29
51.26
53.56
43.44
53.98
54.21
54.96
54.79
55.71
52.52
61.00
53.55
52.70
54.67
54.62
54.30
43.17
53.40
50.14
26.94
39.82
36.04
39.84
118.98
118.35
116.92
120.14
107.26
119.52
121.18
120.44
119.41
118.79
121.09
118.62
121.05
119.42
120.60
119.34
119.88
108.83
119.59
122.69
--
14
--
28.20
28.28
27.54
27.46
36.46
38.68
30.02
39.68
38.85
30.26
28.20
28.37
-28.78
38.93
46
--
--
--
--
--
47
48
49
50
51
118.90
119.40
109.10
108.11
121.40
8.51
8.13
8.30
8.21
8.11
54.76
53.27
43.35
43.00
51.99
27.54
--30.35
118.90
119.40
109.10
108.11
121.40
52
--
-
--
--
--
53
54
55
56
121.41
122.29
122.31
120.06
8.42
8.42
8.41
8.55
53.65
53.79
53.87
48.97
28.70
28.70
31.01
36.54
121.41
122.29
122.31
120.06
--
57
--
--
--
--
--
58
59
60
61
117.64
119.90
108.11
121.05
8.63
7.98
8.29
8.00
55.00
55.44
43.69
54.69
26.30
27.79
-40.01
117.64
119.90
108.11
121.05
--
--
--
--
62
63
64
.
.
-.
.
.
.
.
.
208
65
.
.
66
67
68
69
.
.
.
.
.
.
.
70
....
.
.
.
.
.
.
.
.
.
71
72
122.40
122.04
73
--
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
8.08
8.04
--
52.77
52.47
16.36
16.38
122.40
122.04
--
-
--
....
76
7.90
8.05
7.88
8.16
8.32
8.26
8.23
8.20
8.24
8.50
8.20
8.49
8.48
8.46
8.57
8.10
8.27
8.49
8.25
117.81
107.95
118.46
119.90
108.36
120.23
119.97
120.23
120.78
109.45
119.90
108.97
117.44
120.42
108.60
122.40
117.35
119.88
107.80
--
.
.
.
.
.
.
.
.
.
.
--
--
--
--
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
96
97
98
99
100
101
102
.
.
76
. ....
74
--
Appendix
--
.
.
.
.
.
.
.
----
60.90
43.79
54.03
54.71
43.52
54.91
54.24
54.07
54.16
43.08
54.11
43.09
53.29
53.55
43.97
55.41
58.47
56.57
44.73
--
-30.38
30.43
-27.87
-28.27
28.61
-30.84
-26.80
38.60
-40.09
35.55
26.05
--
--
.
.
117.81
107.95
118.46
119.90
108.36
120.23
119.97
120.23
120.78
109.45
119.90
108.97
117.44
120.42
108.60
122.40
117.35
119.88
107.80
-
----
.
.
.
.
.
.
.
--
--
--
103
114.15
8.01
58.31
61.06
104
--
--
--
--
--
105
106
107
108
109
110
111
112
119.24
121.76
116.47
115.40
117.54
118.60
122.23
120.24
8.73
7.91
7.88
8.13
7.91
8.06
8.00
7.89
54.84
52.42
53.98
54.76
54.17
53.93
50.03
50.44
37.94
16.31
40.09
25.81
30.35
27.13
17.39
39.43
119.24
121.76
116.47
115.40
117.54
118.60
122.23
120.24
113
114
---
---
---
---
---
115
126.07
7.97
54.90
29.36
126.07
Table A4:Assignmt ofTCR
114.15
bd toLMPG-nia& at 310K
209
Appendix
210
Appendix
Publications
1. Duchardt, E.; Richter, C; Reif, B.; Glaser, S. J.; Engels, J. W.; Griesinger, G.; Schwalbe, H
(1999)"Measurement of nJ(H,)-Coupling
Constants by Selective HC(C)H-TOCSY in
Uniformly 13CLabeled RNA",J. Biwd NMR 21,117-126.
2.
Waser, M.; Rueping, M.; Seebach, D.; Duchardt, E.; Schwalbe, H (2001) "On the solution
structure of PHB - Preparation and NIMvRAnalysis of Isotopically Labeled Oligo ((R)-3hydroxybutanoic acids) (OHBs)", Hdv Cim Acta 84, 1821-1845.
3.
Gee, P. J.; Hamprecht, F. A.; Schuler, L. D.; Van Gunsteren, W. F.; Duchardt, E.;
Schwalbe, HI.;Albert, M.; Seebach, D. (2002) "A molecular dynamics simulation study of
the conformational preferences of oligo-(3-hydroxyalcanoic acids) in chloroform
solution.", Hdv Cbim Acta 85, 618-632.
4.
Albert, M.; Seebach, D.; Duchardt, E.; Schwalbe, H (2002) "Synthesis and NMR Analysis
in Solution of Oligo(3-hydroxyalkanoates) with the Side Chains of Alanine, Valine, and
Leucine - Coming Full Circle from PHB to -Peptides to PHB.", Hdv Cim ACa 85, 633658.
5.
Klein-Seetharaman, J.; Oikawa, M.; Grimshaw, S. B.; Winrmer, J.; Duchardt, E.; Ueda, T.;
Imoto, T.; Smith, L. C; Dobson, C M; Schwalbe, H (2002) "Long-range interactions
within a non-native protein", Sdexe 265, 1917-1921.
6.
Duchardt, E.; Richter, C.; Ohlenschliger,
(2004) "Determination of angle
.; G6rlach, M; Wthnert, J.; Schwalbe, H
in RNA from cross-correlated relaxation of CH-dipolar
coupling and N-chemical shift anisotropy" J.Am (Cem Sc 126, 1962-1970.
7.
Duchardt,
E.; Schwalbe, FL (2004) "Residue specific dynamic investigation
cUUCGgtetraloop motif by NMR
3C
relaxation"J. Am
211
man
Sc , submitted.
of the