NMR Dynamics based on chemical
exchange and Hydrogen Exchange
• NMR dynamic methods
• Applications for monitoring calcium signaling
• Detecting intermediates and excited states
– EXSY, CPMG, RDC/PRE
• Amide exchange and application to proteins
and protein folding
• Developing MRI contrast agent
Ca2+ Homeostasis and Signaling
([Ca2+]e 10–3 M)
Ca2+ channels
GPCR
E
NCX
STIM1
RTK
IP3R
RyR
SERCA
SERCA
([Ca2+]i 10-7- 10–6 M)
MCU/MiCa
Letm1
Ca2+
IP3R
Uniporter
RyR
CaM
NCX/HCX
Genes transcription
(NFAT, CREB, DREAM)
[Ca2+]I activated
Buffers
Buffers
Effectors
Ca2+
Ca2+iOscillation frequency
10-6
μs
10-3
ms
1
s
103
min
106
h
The Connexin Family Tree
3
Söhl et al., Nat. Rev. Neurosci. 6: 191-200, 2005
Identifying CaM Binding Region in Gap
Junction Connexins
Score
NH2
COOH
789999999999999876
Cx50_m
145 TKKFRLEGTLLRTYVCHI 162
789999999999987654
Cx46_h
136 RGRVRMAGALLRTYVFNI 153
899999999999766543
Cx44_s
133 RGKVRIAGALLRTYVFNI 150
479999999999999999
Cx43_h
142 HGKVKMRGGLLRTYIISI 161
000999999999999999
CaMKI_h
294 FAKSKWKQAFNATAVVRH 315
000999999999999999
CaMKII_h
289 NARRKLKGAILTTMLATR 310
xxBxB#xxx#xxxx#xxx
1
5
10
Y. Zhou. JJ Yang JBC. 2007; Zhou Y, Yang JJ. Biophys J. 2009 ; Y. Chen, .. JJ Yang Biochem J. 2011
Monitoring Cx Peptide and Calmodulin
[Cx44]/[CaM]
[Cx43]/[CaM]
0:1
0.4:1
0.8:1
1.2:1
9.2
9.1
9.0
T117
0.6:1
0.2
0.9:1
ppm-H
K94
D64
G33
G61
G25
K148
A57
0
1.2:1
-0.1
T29
-0.2
0
0.5
1
1.5
[Cx43]/[CaM]
T117
9.2
1H
9.1
(ppm)
133
115
134
113
132
114
133
115
134
113
132
114
133
115
134
113
132
114
133
115
134
113
132
114
133
115
134
113
132
A57
133
115
2
Y. Zhou. JJ Yang JBC. 2007
A57
114
2.0:1
8.3
132
114
0.3:1
0.1
8.4
113
T29
0:1
8.5
9.0
134
8.5
1H
8.4
(ppm)
5
8.3
Strong Binding Indication by Slow Exchange
CaM :
Cx50p
1:0
Free G33
105.2
105.4
105.6
8.70
8.65
8.60
CaM +
Cx43p
8.55
105.2
1 : 0.4
105.4
105.6
8.70
8.65
8.60
8.55
8.70
8.65
8.60
8.55
8.65
8.60
8.55
105.2
1:1
105.4
105.6
105.2
1:
1.2
105.4
105.6
bound
8.70
6
Y. Chen, .. JJ Yang Biochem J. 2011
HSQC Spectra of Holo-CaM with Cx50 Peptide
Holo-CaM
Holo-CaM + Cx50p
G33
T29
G134
T70
T117
F19
A128
L116
V136
I27
I130
K21
K148
K94
A147
I100
N137
D64
A57
7
Y. Chen, .. JJ Yang Biochem J. 2011
Integration of Calcium Signaling Via CaSR
site5
Site 4
LB1
LB2
Site 1
Site 2
Site 3
Identification of Ca2+
binding sites in ECD of
CaSR
How can CaSR sense
the change of Ca2+o ,
Phe within a narrow
range? (multiple sites?
cooperativity?)
Identification of
CaM binding region
in c-tail of CaSR
Y. Huang, JJ Yang, J Biol Chem. 2007; Yun Huang.. JJ Yang Biochemistry 2009; Y Hang, … JJ Yang, JBC 2010, Zhang, C, 2014
NMR Dynamic Experiments
Initial NMR dynamics experiments in 1970s.
Rapid advancements due to ability to label specific
positions in bio-molecules and methodologies
development
Magnetization exchange spectroscopy (EXSY)-slow
exchange 0.5 s-1 to over 50 s-1
CPMG relaxation dispersion: chemical shifts100~2000s-1,
R1rho can extend to more rapid exchange (dot)
Residual dipolar coupling (RDC) and PRE
Spin relaxation for ns-ps
CPMG and PRE are sensitive to low-lying excited states
with populations > 0.5%
H/D exchange can detect high energy excited states with
much lower population
A.Mittermaier, L. Kay (2009) Trends Biochem. Sci. 34, 601.
A. Mittermaier, L. Kay (2006) Science. 312, 224
K. Wuthrich, G Wagner,(1978) Trends Biochem. Sci. 3, 227
Exchange Spectroscopy (EXSY)
Mittermaier et al (2009) Trends Biochem. Sci. 34, 601.
Slow conformational exchange in the protease ClpP
ClpP, an oligomeric protease comprising 14 subunits with a total molecular mass of 300
kDa. (a) Surface representation of ClpP with two monomers shown as yellow and blue
ribbons. Locations of dynamic isoleucine residues are identified by green and red circles.
Substrate entry pores are indicated with blue arrows. (b) The 1H/13C methyl TROSY
correlation spectrum collected for a uniformly [15N, 2H], Ile δ1 [13C,1H] labeled ClpP
sample. I149 and I151 are each associated with two δ1 methyl peaks, designated F and S,
reflecting slow exchange between two distinct, functionally important, conformations. (50C,
rotational correlational time >0.4 us)
R.Sprangers, PNAS, et al.2005 Wang JM, Cell, 1997
Carr–Purcell–Meiboom–Gill (CPMG) Relaxation Dispersion
100 s-1 ∼ 2000 s-1
Exchange between ground state and excited
state is in the millisecond time scale
kAB
kex = kAB + kBA
A
B
kBA
kBA > kAB
Shape of the dispersion depend on:
• populations of the two states
• chemical shift difference
• the rate of exchange
In a typical series of experiments, variable numbers of refocusing pulses are applied to magnetization as it
evolves under the influence of a chemical shift that varies stochastically due to the exchange process
Baldwin et al (2009) Nature Chem Biol 5, 808.
Minisec Dynamics Relaxation Dispersion of DHFR
Boehr et al (2006) Science 313, 1638.
Relaxation Dispersion (Continue)
Differences in chemical shifts between ground and excited states ()
correlate with differences in peak positions between intermediates (),
suggesting the excited states are similar to the conformation of the
intermediates in the preceding or following step in the catalytic cycle.
Boehr et al (2006) Science 313, 1638.
Residual Dipolar Coupling (RDC)
Mittermaier et al (2009) Trends Biochem. Sci. 34, 601.
Spin Relaxation and Paramagnetic
Relaxation Enhancement
Mittermaier et al (2009) Trends Biochem. Sci. 34, 601.
PRE Study of Maltose Binding Protein
• PRE of holo form agree with Xray structure
• PRE of apo form is larger than
X-ray structure
• The apo form is in a transient
close form (5%)
Grey: N-terminal domain
Blue: C-terminal domain, apo
Red: C-terminal domain, holo
Mittermaier et al (2009) Trends Biochem. Sci. 34, 601.
Hydrogen Exchange Method
Hydrogen exchange (HX) techniques is described
for measuring the approximate exchange rates of
the more labile amide protons in a macromolecule.
The exchangeable amides in proteins are:
Exchangeable
Nucleotides
Hydrogen-Exchange Chemistry
A minimum ~ pH 3.5
> 1hr at pH 3
< 1ms at pH 10
• Hx rate is catalyzed by OH- and H+
kintrinsic
kex = koH [OH-] + kH[H+] + kw
• All exchange rates are referenced
to random coil polyAla at 0 C.
• HX rates are sensitive to pH,local
chemical environment, solvent,
sidechain type, neighboring amino
acids and temperature
• kintrinsic for each amino acid is
different
pD = pH* + 0.4
Bai. And Englander. (1993) Proteins, 17, 75;
Koide S.. and Wright PE J Biomol NMR. 1995 Nov;6(3):306-12.
Simulated Exchange Rates for Labile
Protons of Polypeptides
• In H2O solution at 25 °C.
• Im stands for imidazole
ring NH, Gua for
guanidinium NH, bb for
backbone.
• The amide protons have a
large range of possible
exchange rates under
physiological pH (pH 6.5–
7.5).
Wuthrich &Wagner JMB 1979
HX vs. Protein Structure
In proteins, HX rates can be altered:
H-bonding
Shielding in the center of protein
Shielding by binding another molecules
pH and temperature
Extremely slow exchange can be months,yrs
Protection factor p = kintrinsic/kobs
p > 106-107 for slow exchange
Amide exchange rate contains information
about secondary structural elements
Hx Mechanism (Ex1/EX2)
ko kintrinc
Close p Open
Exchanged
kcl
-Hvidt & Nielsen, 1966
• Solvent penetrates protein secondary structure
• A protected amide hydrogen is ‘closed’ to exchange and
becomes accessible to exchange through an ‘open’ state at the
exchange rate for an unstructured peptide.
Ex1: kintrinc >> kcl
kobs = kop
independent of pH
Ex2: kintrinc << kcl
kobs = kopkintrinc
pH dependent
Ex2 is typically encounted in proteins under conditions where
folded state is stable and intrinsic exchange is relative slow
HX is an excellent way to look at
the stability of proteins
•
The rates of amide proton exchange for individual protons can be
related to equilibrium constants for opening of individual hydrogen
bonds. Knowing the equilibrium constants, one can calculate the free
energy for the conformational transition which allows exchange to
occur.
• When certain protons are only exposed in the completely unfolded
form then the equilibrium constants and Gs correspond to the global
unfolding reaction. These protons are usually the slowest exchanging
protons in the molecule.

GHX = -RT ln(kobs / kintrinc )
•
For mutation, the change of stability:
GHX = (GHX )wt- (GHX )mut =-RT ln (kexwt /kexmut )
Amide Exchange Rates
•Adding D2O to our H2O solution and take spectra at different
times, signals from different amide protons will decrease in
size at different rates. We look at the NH to Ha fingerprint at
different times in DQF-COSY or HSQC.
4.0
t = 0 - No D2O
Add D2O
4.0
(Has)
t = t1
4.0
t = t2
8.0
(NHs)
7.0
Amide Exchange Rates in ACP
• Residues at the center
of helices and
hydrophobic core have
slow exchange rates
Kima Y, et al, BBRC, 2006
• The overall protection
factors (< 10 4.5) are
smaller than other
proteins suggesting that
ACP has high mobility
• Helix II exchanges
faster than helix I and
helix III suggesting that
Helix II is highly
dynamic.
Unfolding/Folding and Misfolding
Competition Hydrogen Exchange
25x dilution
6M GuHCl H2O
pH 7.5
Drop pH 3.8
D2O
Concentrating
1min
Phosphate buffer
pH7.5
NMR
• The refolding experiment involved dilution of droplets of protein
denatured in 6 M GuHCl in H2O solution into a denaturant free
solution of D2O to initiate refolding and hydrogen exchange
simultaneously.
• After folding completed, HX is quenched by lowing the pH.
• Comparing 2D NMR spectrum of the refolded protein with that
was not denatured. Residues protected early in refolding can be
detected using NMR.
Competition HX
of lysozyme
• Using 65 slowly exchanging
amide hydrogen as probes.
The majority of residues in bdomain have exchange >30%.
The majority of residues in adomain have exchange <30%
suggesting that two structural
domains of lysozyme are folding
domains that differ significantly
in the extent to which protected
structure accumulates early in
the folding process.
Miranker et al., Nature, 1991
Pulsed-Label Hydrogen Exchange
•After an adjustable refolding time, tf, the protein is subjected to a
short high pH pulse, where exchange of the unprotected NHs is very
fast. NHs protected by structure within the folding time does not
exchange during the short pulse
•After a pulse time tp. The D to H exchange is quenched by rapidly
lowering the pH.
•After folding completed, the pattern of NH and ND labels in the
refolded protein is analyzed by 2D NMR.
•Increasing tf time, proton occupancies measured in the NMR
spectrum decreases. Plotting proton occupancy vs. folding time tf.
Identifying Folding Pathway
by HX Pulse-Labeling
(a) pure 2-state
All probes achieve 100%
protection at the same rate in
a single kinetic step.
(b) U -> I -> N sequential,
I has A&B H-bonds with the
same HX constant
(c) U1 -> N(30%)
U2 -> N(70%) two
heterogeneous parallel paths
(d) U1-> N
U2->I->N contribution of
intermediate and
heterogeneous folding
Parallel Folding Pathway of Lysozyme
• All probes (50% of 126
amides) have one fast
phase and one slow phase
• Fast phase rates for both aand b domains are 10 ms
• Slow phase rates for a is
65 ms and b is 350 ms,
respectively.
•
a domains folds before b
domain and different
populations of molecules
folded by kinetically
distinct pathways
Additional Methods for Amide-Water Exchange
•
•
•
•
•
•
•
•
Hwang TL, Mori S. Shaka, AJ, and van Zijl PC, Application of Phase-Modulated CLEAN
Chemical EX-change Spectroscopy (CLEANEX-PM) to detect water-protein proton exchange
and intermolecular NOEs. JACS, 1997, 119,6203-6204.
Hwang TL, van Zijl PC, Mori S. Accurate quantitation of water-amide proton exchange rates
using the phase-modulated CLEAN chemical EXchange (CLEANEX-PM) approach with a FastHSQC (FHSQC) detection scheme.J Biomol NMR. 1998 Feb;11(2):221-6.
Clean SEA-HSQC: a method to map solvent exposed amides in large non-deuterated proteins
with gradient-enhanced HSQC J Biomol NMR 2002 Aug;23(4):317-22
Mori S, Abeygunawardana C, Johnson MO, van Zijl PC. Improved sensitivity of HSQC spectra
of exchanging protons at short interscan delays using a new fast HSQC (FHSQC) detection
scheme that avoids water saturation. J Magn Reson B 1995 Jul;108(1):94-8 Erratum in: J Magn
Reson B 1996 Mar;110(3):321
Bougault C, Feng L, Glushka J, Kupce E, Prestegard JH. Quantitation of rapid proton-deuteron
amide exchange using hadamard spectroscopy. J Biomol NMR. 2004 Apr;28(4):385-90.
Miranker A., Robinson CVm Radford, SE, Aplin RT, Dobson CM. Detection of transient protein
folding populations by mass spectrometry. Science. 1993 Nov 5;262(5135):896-900.
Carulla N, Caddy GL, Hall DR, Zurdo J, Gairi M, Feliz M, Giralt E, Robinson CV, Dobson CM.
Molecular recycling within amyloid fibrils.Nature. 2005 Jul 28;436(7050):554-8.
Feng L, Orlando R, Prestegard JH. Mass spectrometry assisted assignment of NMR resonances in
15N labeled proteins.J Am Chem Soc. 2004 Nov 10;126(44):14377-9.
Macnaughtan MA, Kane AM, Prestegard JH. Mass spectrometry assisted assignment of NMR
resonances in reductively 13C-methylated proteins.
J Am Chem Soc. 2005 Dec 21;127(50):17626-7.
CLEANEX-PM spin-locking sequence:
135°(x) 120° (-x) 110° (x) 110°(-x) 120°(x) 135° (-x)
CLEANX-PM has the ability to specifically monitor water-proton
exchange without 1) exchange relayed NOE/ROE from rapidly
exchanging protons (hydroxyl or amide groups) in the
macromolecules, 2) intra-molecular NOE/ROE peaks from protein
CaH protons which has chemical shifts coincident with water, or
TOCSY-type interactions.
FHSQC
CLEANEX-PM
The FHSQC indicates proton signal that remain at the amide
resonance through out the pulse sequence.
The CLEANEX-PM indicates 1H signals that initiate in the
1H O resonance and then transfer to 1H amide resonance
2
during the mixing period of the pulse sequence.
Staphylococcal nuclease –Hwang et al., 1998
Conventional [1H,15N]-HSQC
spectrum of human ubiquitin.
A 0.5 mM15N-labeled sample was prepared in 50 mMpotassium phosphate buffer in
H2O/D2O 5/95, pH=6.2 600 MHz with cryoprobe. it required approximately 21 min
using 128 t1 time increments and 4 scans per increment.
Reconstructed Hadamard [1H,15N]HSQC Spectra for Ubiquitin
(A) Data in 1H2O collected with 128 t1 increments in 20 min. The
sample was then lyophilized overnight and brought back to its initial volume
with 99.9% 2H2O and immediately returned to the spectrometer for rapid
collection of a series of Hadamard spectra.
(B) First point after 1 min in 2H2O collected with 4 scans in 42 s.
Cross-peak intensities as a function of time
Lines are best fits to
I(t) =Io(exp(-kt)+const).
The precision of the data is quite
high with the estimated errors for
rates in the range of 1 × 10-3 min1 being on the order of 5%. Rates
derived also show reasonable
agreement with previously
published rates.
Intermediates Determined by H/D Exchange
Englander (2005) PNAS 102, 4741.
Limitations of Clinically approved Contrast
agents
•
•
•
Paramagnetic
nanoparticles
•
Low relaxivity and requires high dose
injection (0.3-0.5 M)
Low sensitivity, and low
accuracy/specificity
Low S/N, Conc> 0.10 mM
No capability for biomarkers at uM~
nM for molecular imaging
Low resolution
– Detection size > 1-2 cm
– Non-ideal PK/PD with limited
MRI window required for high
quality imaging
Gd Toxicity/Blackbox warning
There is a strong need to develop MRI contrast agents with high
relaxivity, optimized in vivo retention time, organ preference, and
targeting capability.
Lauffer et al, 1987, Xue et al., 2013, 2014
Contrast Agents vs. relaxation
i
ii
Kex
Gd
Rf
Rf
Rs
Kex
Gd
Kex
Gd
Rf
1
cq

T1 T1M   M
1
1
1
1



 c T1e  m  R
Current CAs
A major barrier to the application of MRI technique is its low sensitivity and contrast
limited by the fast rotational correlation time of the molecule (ps).
Macromolecule generated by either covalent binding or the non-covalent nature of the binding
between the monomeric agent and the macromolecules has the improvement in proton
relaxivity is much less than the expected increase based on the molecular-weight increase
due to high internal mobility of the paramagnetic moiety and limited water exchange rate.
ProCAs developed by protein design with increased relaxivity by controlling relaxation
and the capability of targeting specific molecular entities such as cancer biomarkers.
Yang, JJ, JACS, 2010 , Wei et al., 2009, Qiao et al., 2011
Increase Both R1 and R2 by Protein Design
90
r2, ProCA32
r2,
ProCA
1.4 T 3 T 7 T
Relaxivity (mM-1 s-1)
r1, ProCA32
r1,
ProCA
r2, Gd-DTPA
r2,
Clinical
60
r1, Gd-DTPA
r1,
Clinical
30
1
cq
1
1
1
1




T1 T1M   M  c T1e  m  R
1
cq 2nd
 2nd
T12nd T1m
  m2 nd
0
0.01
1
100
10000
Larmar frequency (MHz)
Protein-based Contrast Agents
(ProCAs)
2 2 2
 2
7 2nd
3 2nd
1
2  I g B
0
c
2
c1

S ( S  1)(
) [

]
2nd
2
nd
6
2
nd
2
15
4

T
(r
)
(1   
)
(1    2nd ) 2
1m
GdH
s c2
I c1
At 20 MHz with rGdH2nd=5 Å and q=6, the
second sphere relaxivity could potentially
ProCA1 as a MRI contrast agent
A
H2O
PEG
H2O
D 62
E 15
D 58 Gd
K 66
D 56 D 64
K 45
9.05 ns
9.20 ns
K 47
PEG
PEG
•q=2.0
q
=
1.
8
Sample
Gd3+
Zn2+
Ca2+
Mg2+
Log
(KGd/KZn)
Log
(KGd/KCa)
Log
(KGd/KMg)
DTPA28
22.45
18.29
10.75
18.20
4.17
11.70
4.25
DTPABMA41
16.85
12.04
7.17
na*
4.81
9.68
na*
CA1.CD2
12.06
6.72
<2.22
<2.0
5.34
>9.84
>10.06
45
HER2targeted ProCA Detects Breast and Ovarian
Cancers depending on HER2 Expression Levels
Pre
24 hr
30 min
MDA-MB-231
SKOV-3
Intensity Enhancement
24 hr
24 hr
40
Blood Vessel
Positive tumor stained
with ProCA1-affi-m
Positive tumor stained
with HER2 antibody
4 hr
4 hr
Enhancement %
Blood Vessel
SKOV-3
MDA-MB-231
30
20
10
0
5 min 30 min
Positive tumor stained
with ProCA1-affi-m
Positive tumor stained
with HER2 antibody
3 hr
24 hr
52 hr
Time points
Excellent Tumor Penetration And Distribution, Superior Than
Protein-based Contrast Agents (ProCAs)
A
H2O
PEG
H2O
D 62
E 15
D 58 Gd
K 66
D 56 D 64
K 45
K 47
PEG
PEG
GRPR, GPCR
• First computational design of Gd3+ -binding site in proteins as novel protein
MRI reagents with strong metal stability and selectivity
• 10-20 fold in vitro increases in R1 and R2, high R1 at 7T
• 100 fold increase in in vivo detection resolution. Extend to earlier and
accurate detection of liver lesion size from > 2 cm to <0.25 mm
micrometstasis by dual ratiometric imaging
• ProCAs increase specificity by enabling molecular imaging of breast and
prostate cancer biomarkers Her2/EGFR and GRPR with enhanced sensitivity,
optimized retention time, and proper tissue/tumor penetration
• 20-100 fold low dose injection, biocompatible, no acute toxicty
Xue, 2013, 2014, Qiao, 2014, L. Wei et al., 2010, Fan Pu unpublished