Structural Analysis of Protein
Structure
Circular Dicroism
Fluorescence
X-ray
NMR
Methods for Secondary Structural Analysis
• A number of experimental techniques can selectively
examine certain general aspects of macromolecular
structure with relatively little investment of time and
sample.
• Reasonable estimates of protein secondary structure
content can be determined empirically through the use of
Circular dichroism (CD) spectroscopy
Nuclear Magnetic Resonance (NMR) spectroscopy
FT-infrared spectroscopy
Circular Dichroism
• Circular dichroism (CD) spectroscopy is a form of light
absorption spectroscopy that measures the difference in
absorbance of right- and left-circularly polarized light (rather
than the commonly used absorbance of isotropic light) by a
substance.
• It is measured with a CD spectropolarimeter. The instrument
needs to be able to measure accurately in the far UV at
wavelengths down to 190 - 170 nm (170 - 260 nm).
• The difference in left and right handed absorbance A(l)- A(r)
is very small (usually in the range of 0.0001) corresponding to
an ellipticity of a few 1/100th of a degree.
Physics of
CD
• Linear polarized light can be viewed as a superposition of
opposite circularly polarized light of equal amplitude and phase.
• A projection of the combined amplitudes perpendicular to the
propagation direction thus yields a line.
• When this light passes through an optically active sample with a
different absorbance A for the two components, the amplitude
of the stronger absorbed component will be smaller than that of
the less absorbed component. The consequence is that a
projection of the resulting amplitude yields an ellipse instead of
the usual line, while the polarization direction has not changed.
The occurrence of ellipticity is called Circular Dichroism.
Rotation of Plane-polarized Light by
an Optically Active Sample
• Pockels cell produces a beam that is alternately switched
between L and R. The beam then passes through the
sample to a photomultiplier. The detected signal can then
be processed as ΔA vs λ.
Physical Principles of CD
• Inherently asymmetric chromophores (uncommon) or
symmetric chromophores in asymmetric environments will
interact differently with right- and left-circularly polarized
light resulting in circular dichroism.
• Right- and left-circularly polarized light will be absorbed
to different extents at some wavelengths due to differences
in extinction coefficients for the two polarized rays called
circular dichroism (CD).
• Circular dichroism can only occur within a normal
absorption band and thus requires either an inherently
asymmetric chromophore (uncommon) or a symmetric one
in an asymmetric environment.
Instrumentation
• The most common instruments around are the
currently produced JASCO, JobinYvon, OLIS, and
AVIV models.
• We have the Jasco 710 and 810 models with
temperature controllers. The air cooled 150W
Xenon lamp does not necessitate water cooling.
• You still need to purge with ample nitrogen to get
to lower wavelengths (below 190 nm).
Typical Initial Concentrations
• Protein Concentration: 0.5 mg/ml (The protein concentration
needs to be adjusted to produce the best data).
• Cell Path Length: 0.5-1.0 mm. If absorption poses a problem,
cells with shorter path (0.1 mm) and a correspondingly increased
protein concentration and longer scan time can be employed.
• Stabilizers (Metal ions, etc.): minimum
• Buffer Concentration: 5 mM or as low as possible, while
maintaining protein stability. A typical buffer used in CD
experiments is 10 mM phosphate, although low concentrations
of Tris, perchlorate or borate is also acceptable.
• As a general rule of thumb, one requires that the total
absorbance of the cell, buffer, and protein be between 0.4 and
1.0 (theoretically, 0.87 is optimal).
• A spectra for secondary structure determination (260 - 178 nm)
will require 30-60 minutes to record (plus an equivalent amount
of time for a baseline as every CD spectrometer.
Sample Preparation and Measurement
• Additives, buffers and stabilizing compounds: Any compound,
which absorbs in the region of interest, (250 - 190 nm) should be
avoided. A buffer or detergent, imidazole or other chemical should
not be used unless it can be shown that the compound in question
will not mask the protein signal.
• Protein solution: The protein solution should contain only those
chemicals necessary to maintain protein stability/solubility, and at
the lowest concentrations possible. The protein itself should be as
pure as possible, any additional protein will contribute to the CD
signal.
• Contaminants: Particulate matter (scattering particles), anything
that adds significant noise (or artificial signal contributions) to the
CD spectrum must be avoided. Filtering of the solutions (0.02 m
syringe filters) may improve signal to noise ratio.
• Data collection: Initial experiments are useful to establish the best
conditions for the "real" experiment. Cells of 0.5 - 1.0 mm path
length offer a good starting point.
CD Data Analysis
• The difference in absorption to be measured is very small.
The differential absorption is usually a few 1/100ths to a
few 1/10th of a percent, but it can be determined quite
accurately. The raw data plotted on the chart recorder
represent the ellipticity of the sample in radians, which can
be easily converted into degrees
CD Data Analysis
• To be able to compare these ellipticity values we need to
convert into a normalized value. The unit most commonly
used in protein and peptide work is the mean molar
ellipticity per residue. We need to consider path length l,
concentration c, molecular weight M and the number of
residues.
in proper units (CD spectroscopists use decimol)
which finally reduces to
The values for mean molar ellipticity
per residue are usually in the 10,000's
CD Data Analysis
• The molar ellipticity [] is related to the difference in
extinction coefficients
Δε [] = 3298 Δε.
• Here [] has the standard units of degrees cm2 dmol -1
• The molar ellipticity has the units degrees deciliters
mol-1 decimeter-1.
Circular Dichroism of Proteins
• It has been shown that CD spectra between 260 and
approximately 180 nm can be analyzed for the different
secondary structural types: alpha helix, parallel and antiparallel beta sheets, turns, and other.
• A number of excellent review articles are available
describing the technique and its application (Woody, 1985
and Johnson, 1990).
• Modern secondary structure determination by CD are
reported to achieve accuracies of 0.97 for helices, 0.75 for
beta sheet, 0.50 for turns, and 0.89 for other structure types
(Manavalan & Johnson, 1987).
CD Signal of Proteins
• For proteins we will be mainly concerned with absorption
in the ultraviolet region of the spectrum from the peptide
bonds (symmetric chromophores) and amino acid
sidechains in proteins.
• Protein chromophores can be divided into three classes: the
peptide bond, the amino acid sidechains, and any
prosthetic groups.
• The lowest energy transition in the peptide chromophore is
an n → p* transition observed at 210 - 220 nm with very
weak intensity (emax~100).
----p* p → p* ~`190 nm emax~7000
----n n → p 208-210, 191-193 nm emax~100
----p
Comparison of the
UV absorbance
(left) and the
circular dichroism
(right) of poly-Llysine in different
secondary structure
conformations as a
function of pH.
• The n → p* transition appears in the a-helical form of the
polymer as a small shoulder near 220 nm on the tail of a much
stronger absorption band centered at 190 nm. This intense band,
responsible for the majority of the peptide bond absorbance, is a
p → p* transition (emax ~ 7000).
• Using CD, these different transitions are more clearly evident.
Exciton splitting of the p → p* transition results in the negative
band at 208 and positive band at 192 nm.
CD Spectra of Proteins
• Different secondary structures of peptide bonds have
different relative intensity of n → p* transitions, resulting
in different CD spectra at far UV region (180 - 260 nm).
• CD is very sensitive to the change in secondary structures
of proteins. CD is commonly used in monitoring the
conformational change of proteins.
• The CD spectrum is additive. The amplitude of CD curve
is a measure of the degree of asymmetry.
• The helical content in peptides and proteins can be
estimated using CD signal at 222 nm
e222= 33,000 degrees cm2 dmol -1 res-1
• Several curve fitting algorithms can be used to deconvolute
relative secondary structures of proteins using the CD
spectra of proteins with known structures.
Protein CD Signal
• The three aromatic side chains that occur in proteins (phenyl
group of Phe, phenolic group of Tyr, and indole group of
Trp) also have absorption bands in the ultraviolet spectrum.
However, in proteins, the contributions to the CD spectra in
the far UV (where secondary structural information is
located) is usually negligible. Aromatic residues, if
unusually abundant, can have significant effects on the CD
spectra in the region < 230 nm, complicating analysis.
• The disulfide group is an inherently asymmetric
chromophore as it prefers a gauche conformation with a
broad CD absorption around 250 nm.
[] x10-3 degrees cm2 dmol -1
Far UV CD Spectra of Proteins
• Each of the three
basic secondary
structures of a
polypeptide chain
(helix, sheet, coil)
show a characteristic
CD spectrum. A
protein consisting of
these elements should
therefore display a
spectrum that can be
deconvoluted into the
three individual
contributions.
CD Spectra of
Protein
CD Spectra Fit
• In a first approximation, a CD spectrum of a protein or
polypeptide can be treated as a sum of three components:
a-helical, b-sheet, and random coil contributions to the
spectrum.
• At each wavelength, the ellipticity (θ) of the spectrum will
contain a linear combination of these components:
(1)
•
θT is the total measured susceptibility, θh the contribution
from helix, θs for sheet, θc for coil, and the corresponding
χ the fraction of this contribution.
CD Spectra Fit
• As we have three unknowns in this equation, a
measurement at 3 points (different wavelengths) would
suffice to solve the problem for χ, the fraction of each
contribution to the total measured signal.
• We usually have many more data points available from our
measurement (e.g., a whole CD spectrum, sampled at 1 nm
intervals from 190 to 250 nm). In this case, we can try to
minimize the total deviation between all data points and
calculated model values. This is done by a minimization of
the sum of residuals squared (s.r.s.), which looks as
follows in our case :
Using CD to Monitor 3º Structure of Proteins
• CD bands in the near UV region (260 – 350 nm) are
observed in a folded protein where aromatic sidechains are
immobilized in an asymmetric environment.
• The CD of aromatic residues is very small in the absence of
ordered structure (e.g. short peptides).
• The signs, magnitudes, and wavelengths of aromatic CD
bands cannot be calculated; they depend on the immediate
structural and electronic environment of the immobilized
chromophores.
• The near-UV CD spectrum has very high sensitivity for the
native state of a protein. It can be used as a finger-print of
the correctly folded conformation.
Domain 1of CD2
CD2 is a cell adhesion
molecules.
Domain 1 of CD2 has a IgG
fold. Nine b-strands form a
beta-sandwich structure.
Two Trp residues, W-7 and
W-32 (green) are located at
the exposed and buried
region of the protein,
respectively.
Our lab has used CD2 as a
model system to understand
conformation flexibility of
proteins
CD2 is Stable from pH 1 to 10
10 00
0
-50 0
-10 00
[] (deg cm
2
dmol
-1
res
-1
)
50 0
-15 00
-20 00
-25 00
-30 00
20 0
21 0
22 0
23 0
Wavelength (nm)
24 0
25 0
26 0
Conformational Change of CD2
c
0
-1
[ ] (deg cm2 dmol )
6M GuHCl
-1000
25 ºC
-2000
85 ºC
-3000
200
210
220
230
Wavelength (nm)
240
250
260
CD2 Becomes Significantly Helical in TFE
[ ] (deg cm2 dmol -1 res -1)
50 00
0
-50 00
0% T FE
10 % T FE
17 % T FE
19 % T FE
30 % T FE
80 % T FE
4
-1 10
-1.5 1 04
-2 104
20 0
21 0
22 0
23 0
Wavelength (nm)
24 0
25 0
26 0
Near UV CD Spectra of CD2
200
• CD2 losses
its native
well packed
tertiary
structure at
high
temperature
and in 6M
GuHCl
a
6 MGuHCl
85 ºC
0
2
[ ] (deg cm dmol -1 )
100
-100
-200
25 ºC
-300
-400
260
280
300
Wavelength (nm)
320
340
360
CD2 losses its Tertiary Structure in TFE
2 00
-1
-1
[ ] (deg cm2 dmol res )
1 00
0
-10 0
0 % TFE
1 0% T FE
-20 0
1 7% T FE
3 0% T FE
-30 0
-40 0
2 60
2 70
2 80
2 90
Wavelength (nm)
3 00
3 10
3 20
Trp Fluorescence Emission Spectra of
CD2 under Different Conditions
c
4
4 10
Trp
Fluorescence intensity
25ºC
4
3 10
4
2 10
85ºC
4
1 10
0
300
320
340
360
Wavelength (nm)
380
400
6M GuHCl
• In a hydrophobic
environment (inside of
a folded protein), Trp
emission occurs at
shorter wavelength.
When it is exposed to
solvent, its emission is
very similar to that of
the free Trp amino acid
(red shift occurs).
Secondary Structure Prediction of CD2
x-structure
A
1
B
10
20
PHD
GOR
SOPMA
x-structure
D
E
60
PHD
GOR
SOPMA
30
C'
40
C"
50
RDSGTVWGALGHGINLNIPNFQMTDDIDEVRWERGSTLVAEFKRKMKPFLK
CCCCSSSSCCCCCSSSCCCCCCCCCCHHHHHHHHCCHHHHHHHHHCCCCSS
CCCCSSSSSSSCCCSCCCCCCCCCCCHCHSSHHHCCHHHHHHHHHHHHHHH
CCCCSSHCCCCCCSSSCCCCCCCCCCCCHSSHHCCCSHHHHHHHHHHHHHC
Rat CD2
Rat CD2
C
F
70
G
80
90
SGAFEILANGDLKIKNLTRDDSGTYNVTVYSTNGTRILNKALDLRILE
CCCSSSSSCCCSSSCCCCCCCCCCSSSSSSCCCHHHHHHHHCCCCCCC
HHHHHHHHHHHHHHHSSSSCCCCSSSSSSSSCCCCSSHHHHHHHHHHH
CCCSSSSCCCCSSSSSSCCCCCCCSSSSSSSCCCCSSSSHHHHHSSHC
H = a-helix
b-sheet
S = b-sheet
C = coil
310-helix
CD2 vs. Helical Propensity
• Residues on strands
C, C’, C” and G
have strong helical
propensity.
F42
C"
C
C'
F
D
V78
E
G
W3 2
B
L1 6
V39
Y76
A
C
N
Summary of CD
• Circular dichroism spectroscopy is used to gain information
about the secondary structure and folded state of proteins and
polypeptides in solution.
• Benefits: Uses very little sample (200 ul of 0.5 mg/ml solution
in standard cells)
Non-destructive
Relative changes due to influence of environment on
sample (pH, denaturants, temperature, etc.) can be
monitored accurately.
• Drawbacks: Interference with solvent absorption in the UV
region
Only very dilute, non-absorbing buffers allow
measurements below 200 nm
Absolute measurements subject to a number of
experimental errors
Average accuracy of fits about +/- 10%
CD spectropolarimeter is relatively expensive
X-ray Crystallography
• X-rays are electromagnetic radiation at
short wavelengths, emitted when electrons
jump from a higher to a lower energy state.
–
–
–
–
–
Growth of crystals
X-ray diffraction
Heavy-metal complex
Build model
Refinement
Drug design
information
Crystallization
X-ray
crystallography
Structure
analysis
Model refinement
Data collection
Data procession
http://www-structure.llnl.gov/xray/101index.html; http://www.aps.anl.gov/aps/frame_home.html
Crystal
• A crystal is built up from many billions of small identical units, or unit
cells. These unit cells are packed against ach other in three dimensions,
much as identical boxes are packed and stored in a warehouse. The unit
cell may contain one or more than one molecule. Although the number of
molecules per unit cell is always the same for all the unit cells of a single
crystal, it may vary between different crystal forms of the same protein.
The diagram shows in two dimensions several identical unit cells, each
containing two objects packed against each other. The two objects within
each unit cell are related by twofold symmetry to illustrate that each unit
cell in a protein crystal can contain several molecules that are related by
symmetry to each other.
Many small identical blocks or unit cells
are packed against other in 3D.
In order to obtain a crystal, molecules
must assemble into a periodic lattice.
Each unit cell can contain
several molecules that are
related by symmetry.
The diagram shows identical
blocks, each containing two
objects packed against each
other.
www.via.ecp.fr/~im/musee/escher.html
Crystals & X-ray Diffraction
enzyme RuBisCo
• Well-ordered protein crystals (a) diffract x-rays and produce
diffraction patterns that can be recorded on film (b) (Laue
photograph). The diffraction pattern was obtained using
polychromatic radiation from a synchrotron source in the
wavelength region 0.5 to 2.0 Å.
Protein Crystal Packing
• Protein crystals contain large
channels and holes filled with
solvent molecules. The
subunits (colored disks) form
octamers of molecular weight
around 300 kDa of glycolate
oxidase, with a hole in the
middle of each of about 15 Å
in diameter. Between the
molecules there are channels
(white) ~ 70 Å in diameter
through the crystal.
The Hangingdrop Method of
Protein
Crystallization
• About 10 ml of a 10 mg/ml protein solution in a buffer with added
precipitant --- such as ammonium sulfate, at a concentration below that at
which it causes the protein to precipitate --- is put on a thin glass plate that
is sealed upside down on the top of a small container. In the container
there is about 1 ml of concentrated precipitant solution. Equilibrium
between the drop and the container is slowly reached through vapor
diffusion, the precipitant concentration in the drop is increased by loss of
water to the reservoir, and once the saturation point is reached the protein
slowly comes out of solution. If other conditions such as pH and
temperature are conducive, protein crystals will form in the drop.
A Diffraction Experiment
When the X-ray goes through the crystal, beams is diffracted and diffraction
pattern is recorded on a detector. The crystal is rotated a certain degree while
this pattern is recorded. A series of frames are collected.
Determine the size of the unit cell by Bragg's law:
2dsin = λ
d= λ/(2* sin).
http://www-structure.llnl.gov/Xray/101index.html
A Diffraction Experiment
• A narrow beam of x-rays (red) is
taken out from the x-ray source
through a collimating device. When
the primary beam hits the crystal,
most of it passes straight through, but
some is diffracted by the crystal.
These diffracted beams, which leave
the crystal in many different
directions, are recorded on a detector,
either a piece of x-ray film or an area
detector. The crystal was rotated one
degree while this pattern was
recorded. The pattern of RuBisCo
was collected using polychromatic
radiation.
Diffraction of Xrays by a Crystal
• (a) When a beam of x-rays (red) shines
on a crystal all atoms in the crystal
scatter x-rays in all directions. Most
of these scattered x-rays cancel out,
but in certain directions (blue arrow)
they reinforce each other and add up to
a diffracted beam. Different sets of
parallel planes (b) can be arranged
through the crystal so that each corner
of all unit cells is on one of the planes
of the set. X-ray diffraction can be
regarded as reflection of the primary
beam from sets of parallel planes in
the crystal, separated by a distance d.
The primary beam strikes the planes at
an angle  and the reflected beam
leaves at the same angle, the reflection
angle.
Diffraction of
X-rays by a
Crystal
• X-rays (red) that are reflected from the lower plane have traveled
farther than those from the upper plane by a distance BC + CD, which
is equal to 2dsin.
• Reflection can only occur when this distance is equal to the wavelength
l of the x-ray beam and Bragg's law (2dsin = l). To determine the
size of the unit cell, the crystal is oriented in the beam so that reflection
is obtained from the specific set of planes in which any two adjacent
planes are separated by the length of one of the unit cell axes. This
distance, d, is then equal to l/(2sin). The wavelength, l, of the beam
is known since we use monochromatic radiation. The reflection angle,
, can be calculated from the position of the diffracted spot on the film,
where the crystal to film distance can be easily measured. The crystal
is then reoriented, and the procedure is repeated for the other two axes
of the unit cell.
Diffraction of
X-ray Beams
• The reflection angle, q, for a
diffracted beam can be calculated
from the distance (r) between the
diffracted spot on a film and the
position where the primary beam
hits the film. From the geometry
shown in the diagram, the tangent
of the angle 2 = r/A. A is the
distance between crystal and film
that can be measured on the
experimental equipment, while r
can be measured on the film.
Hence,  can be calculated. The
angle between the primary beam
and the diffracted beam is 2, as
can be seen on the enlarged insert
to the right. It shows that this angle
is equal to the angle between the
primary beam and the reflecting
plane plus the reflection angle, both
of which are equal to .
Properties of Diffracted Waves
• Two diffracted beams, each of which is defined by three properties:
amplitude, which is a measure of the strength of the beam and
which is proportional to the intensity of the recorded spot,
phase, which is related to its interference, positive or negative,
with other beams, and
wavelength, which is set by the x-ray source for monochromatic
radiation.
• We need to know all three properties to determine the position of the
atoms giving rise to the diffracted beams.
Multiple Isomorphous
Replacement (MIR)
• Heavy atoms (strong diffraction) are introduced into the
unit cell of the crystal to obtain phase information by
soaking crystals in the metal solution.
• Intensity differences are used to deduce the positions of the
heavy atoms in the crystal unit cell. Fourier summations of
these intensity differences give Patterson maps of the
vectors between the heavy atoms.
• From the positions of the heavy atoms in the unit cell, we
can get amplitudes and phases.
• More than two different heavy-metal complexes are
needed to give a reasonably good phase determination for
all reflections.
Building a Model
• The amplitude and phases of the diffraction data from the
protein crystals are used to calculate an electron-densitymap of the repeating unit of the crystal.
• This map is then interpreted as a polypeptide chain with a
particular amino acid sequence.
• The resolution (in Å) is limited by the map error,
resolution of the diffraction map.
• At low resolution (5 Å or higher), the shape of the
molecule can be obtained.
• At medium resolution (~3 Å), the trace of the polypeptide
chain, i.e. active site, can be obtained
• At high resolution ( 2 Å), the a.a. sidechians can be
resolved.
Electron-density maps at different resolution
show more detail at higher resolution.
(d) 1.1 Å
Interpreting Electron-density Maps
• The electron-density map is interpreted by fitting into it pieces of a
polypeptide chain with known stereochemistry such as peptide groups
and phenyl rings. The electron density is displayed on a graphics
screen in combination with a part of the polypeptide chain (red) in an
arbitrary orientation (a). The units of the polypeptide chain can then
be rotated and translated relative to the electron density until a good
fit is obtained (b).
High Resolution
Crystal Structures
F. Liu
Reducing Errors by Refinement
In the refinement process, the model is changed to minimize
the difference between the experimentally observed
diffraction amplitudes and those calculated for a
hypothetical crystal containing the model instead of the real
molecules.
The difference is called the R factor, with 0.0 being exact
agreement and 0.59 total disagreement.
0.15 < R < 0.20 = well determined structure
R ~ 0.30 = medium structure
R > 0.30 = bad structure
B-factor
ATOM
ATOM
ATOM
ATOM
1
2
3
4
N
CA
C
O
PRO
PRO
PRO
PRO
A
A
A
A
190
190
190
190
-0.567
-0.399
-1.288
-2.520
24.363
23.026
21.990
22.007
16.753
17.339
16.644
16.772
49.28
49.21
49.61
49.44
• In the pdb file of x-ray structures, the atoms positions is given
by four numbers, three of them for coordinates and one
quantity B, which is called the B-factor or temperature factor.
• B < 20 = well defined regions
• B > 40 = atoms have high flexibility
NMR Spectroscopy
• It is possible to determine the secondary structure of a protein
using NMR techniques without determining the threedimensional structure. NMR is potentially the most powerful of
all the methods available for prediction of secondary structure.
Unlike secondary structure determinations by CD, which provide
overall secondary structure content (% helix, % sheet, etc.), using
NMR parameters, secondary structures are localized to specific
segments of the polypeptide chain.
• However, obtaining secondary structure from NMR data requires
considerably more material (milligrams) and effort (requires
sequence specific resonance assignments) than the other
spectroscopic techniques and is limited to proteins of molecular
weight amenable to NMR investigation (< 35 - 40 kDa).
NMR Spectroscopy
• In the past 10 years, nuclear magnetic resonance (NMR)
spectroscopy has proved itself as a potentially powerful
alternative to X-ray crystallography for the determination
of macromolecular three-dimensional structure. NMR has
the advantage over crystallographic techniques in that
experiments are performed in aqueous solution as opposed
to a crystal lattice.
• However, the physical principles that makes NMR
possible, limits the application of this technique to
macromolecules of less than 35 - 40 kDa. Fortunately, a
large number of globular proteins and most protein
domains fall into this molecular weight regime.
Physical Principles of NMR
• Sub-atomic particles (e.g., proton, neutron, electron, etc.)
possess a characteristic called spin angular momentum. From
quantum mechanics, each particle has a spin value of 1/2. The
combination of multiple particles in the nucleus results in an
overall spin property for each atomic isotope. Those isotopes
with an even number of protons and neutrons will have zero
magnetic spin (e.g., He-4, C-12 and O-16). An odd number of
protons and an even number of neutrons (e.g., H-1, N-15, or F19) or an odd number of neutrons and an even number of
protons (e.g., He-3, O-17 or Ca-41) result in an overall
(multiple of 1/2) spin. Those isotopes with odd numbers of both
protons and neutrons (e.g., H-2 or N-14) have more complex
spin states and are less suitable for direct NMR observation in
macromolecules.
Physical Principles of NMR
• Fortunately, each of the four most abundant elements in
biological material (H, C, N, and O) have at least one
naturally occurring isotope with non-zero nuclear spin, and
in principle, can be observed using NMR.
• The naturally occurring isotope of hydrogen, H-1, is present
at > 99 % abundance and forms the basis of the experiments
described here. Other important NMR-active isotopes
include C-13 and N-15 present at 1.1 and 0.4 % natural
abundance, respectively. The low natural abundance of these
two isotopes makes their observation difficult on commonly
isolated natural products.
• These two nuclei are however very extensively used for
larger (> 10 kDa) proteins, which can be isotopically
enriched (to > 95 % if necessary) when cloned into systems
with high expression yields.
Chemical Shifts
• In the presence of an external magnetic field, the spin
angular momentum of nuclei with isotopes of overall nonzero spin will undergo a cone-shaped rotation motion called
precession. The rate (frequency) of precession for each
isotope is dependent on the strength of the external field and
is unique for each isotope.
• For example, in a magnetic field of a given strength (e.g.
14.1 Tesla) all protons in a molecule will have characteristic
resonance frequencies (chemical shifts) within a dozen or so
parts per million (ppm) of a constant value (e.g., 600.13
MHz) characteristic of the particular nuclear type.
• These slight differences are due to the type of atom the
proton is bound to (e.g., C, N, O, or S) and the local
chemical environment. Thus each proton should, in
principle, be characterized by a unique chemical shift.
One-dimensional NMR Spectra
• The NMR signals (chemical
shifts) for all the hydrogen atoms
in this small molecule are clearly
separated from each other. In this
spectrum, the signal from the CH3
protons is split into three peaks
and that from the CH2 protons
into four peaks close to each other,
due to the experimental
conditions.
• 1H-NMR spectrum of a small
protein, the C-terminal domain of
a cellulase, comprising 36 amino
acid residues. The NMR signals
from many individual hydrogen
atoms overlap and peaks are
obtained that comprise signals
from many hydrogen atoms.
Chemical Shifts
• Some protons such as the three protons of each sidechain
methyl group of Thr, Val, Leu, Ile, and Met and most pairs
of equivalent (2,6 and 3,5) aromatic ring protons are found
to have degenerate chemical shifts.
• Other protons (e.g., some OH, SH, and NH3) are in rapid
chemical exchange with the solvent, and thus have
chemical shifts indistinguishable from the solvent
resonance. Nevertheless, nearly complete chemical shift
assignments are often possible and are a prerequisite for
structural studies by NMR.
Chemical shifts
• Since the chemical shift of a nucleus is sensitive to the
environment, it should also contain structural information.
• Correlations between chemical shift tendencies and secondary
structures have been identified. The alpha proton of all 20
naturally occurring amino acids has been shown to have a strong
correlation with secondary structure. Wishart et al., (1992) have
produced a simple method for secondary structure determination
by analyzing the difference between the alpha proton chemical
shift for each residue and that reported for the same residue type
in a "random coil" conformation. Helical segments have
groupings of alpha protons whose chemical shifts are
consistently less than the random coil values whereas beta
strands had values consistently greater. In this way, the location
of helix and strand segments are possible (and quite reliable)
although the boundaries of the secondary structural elements are
not as well defined.
Secondary Shifts
• Plot of the
differences
between the
observed alpha
proton chemical
shifts and the
corresponding
random coil
values, d(Hanative)
- d(Harandom),
versus the amino
acid sequence of
Glutaredoxin 3
J coupling
• Structural information from NMR experiments come
primarily from through-bond (scalar or J coupling) or through
space (the nuclear Overhauser effect NOE) magnetization
transfer between pairs of protons.
• J couplings between pairs of protons separated by  three
covalent bonds can be measured. The value of a three-bond J
coupling constant contains information about the intervening
torsion angle. This is called the Karplus relationship and has
the form:
3J = A cos (θ) +B cos2 (θ) + C
where A, B, and C are empirically derived constants for each
type of coupling constant (e.g., 3JHAHN or 3JHAHB).
J coupling
• Shown above is the empirically-derived Karplus relationship
between the vicinal three-bond coupling constant 3JHNa and the
intervening torsion angle phi.
Coupling Constants
• The three-bond coupling constant between the intra-residual
alpha and amide protons is the most useful for secondary
structure determinations as it can be directly related to the
backbone dihedral angle phi.
3J
• right-handed alpha helix, phi = -57º,
HAHN = 3.9 Hz
3J
• right handed 3.10 helix, phi = -60º,
HAHN = 4.2 Hz
3J
• antiparallel beta sheet, phi = -139º,
HAHN = 8.9 Hz
3J
• parallel beta sheet, phi = -119º,
HAHN = 9.7 Hz
3J
• left-handed alpha helix, phi = 57º,
HAHN = 6.9 Hz
Two-dimensional NMR Spectrum
• The peaks along the
diagonal correspond to
the 1D spectrum. The
off-diagonal peaks in
this NOE spectrum
represent interactions
between hydrogen
atoms that are closer
than 5 Å to each other
in space. From such a
spectrum, one can
obtain information on
both the secondary
and tertiary structures
of the protein.
COSY NMR Experiments
•
COSY NMR experiments
give signals that correspond
to hydrogen atoms that are
covalently connected through
one or two other atoms.
Since hydrogen atoms in two adjacent residues are covalently
connected through at least three other atoms (for instance,
HCa-C'-NH), all COSY signals reveal interactions within the same
amino acid residue. These interactions are different for different
types of side chains. The NMR signals therefore give a "fingerprint"
of each amino acid. The diagram illustrates fingerprints (red) of
residues Ala and Ser.
NOE
• NOE NMR experiments give
signals that correspond to
hydrogen atoms that are close
together in space (less than 5
Å), even though they may be
far apart in the amino acid
sequence. Both secondary and
tertiary structures of small
protein molecules can be
derived from a collection of
such signals, which define
distance constraints between a
number of hydrogen atoms
along the polypeptide chain.
NOE
• The other major source of structural information comes
from through space dipole-dipole coupling between two
protons called the NOE. The intensity of a NOE is
proportional to the inverse of the sixth power of the
distance separating the two protons and is usually observed
if two protons are separated by < 5 Å. Thus, the NOE is a
sensitive probe of short intramolecular distances. NOEs
are categorized according to the location of the two protons
involved in the interaction.
• Intraresidual NOEs are between protons within the same
residue, whereas sequential, medium, and long range
NOEs are between protons on residues sequentially
adjacent, separated by 1, 2 or 3 residues, and separated by
four or more residues in the polypeptide sequence. A
network of these short inter-proton distances form the
backbone of three-dimensional structure determination by
NMR.
Sequential
Assignment
• Adjacent residues in the amino acid sequence of a protein
can be identified from NOE spectra. The H atom attached to
residue i + 1 (orange) is close to and interacts with (purple
arrows) the H atoms attached to N, Ca, and Cb of residue i
(light green). These interactions give cross-peaks in the
NOE spectrum that identify adjacent residues and are used
for sequence-specific assignment of the amino acid
fingerprints derived from a COSY spectrum.
NOE
• Regions of secondary structure in
a protein have specific
interactions between hydrogen
atoms in sequentially nonadjacent
residues that give a characteristic
pattern of cross-peaks in an NOE
spectrum. In antiparallel b-sheet
regions there are interactions
between Ca-H atoms of adjacent
strands (pink arrows), between NH and Ca-H atoms (dark purple
arrows), and between N-H atoms
of adjacent strands (light purple
arrows). The corresponding
pattern of cross-peaks in an NOE
spectrum identifies the residues
that form the antiparallel b sheet.
Parallel b sheets and a helices are
identified in a similar way.
NOEs
• A number of short (< 5 Å) distances are fairly unique to
secondary structural elements.
• alpha helices are characterized by short distances between
certain protons on sequentially neighboring residues (e.g.,
between backbone amide protons, dNN, as well as between beta
protons of residue i and the amide protons of residue i+1, dbN.
Helical conformations result in short distances between the
alpha proton of residue i and the amide proton of residues i+3
and to a lesser extent i+4 and i+2. These i+2, i+3, and i+4
NOEs are collectively referred to as medium range NOEs
• NOEs connecting residues separated by more than 5 residues
are referred to as long range. Extended conformations (e.g.,
beta strands) on the other hand, are characterized by short
sequential, daN, distances. The formation of sheets also result
in short distances between protons on adjacent strands (e.g.,
daa and daN).
Amide Proton Exchange Rates
• The regular hydrogen-bonded secondary structures "protect"
amide protons involved in them as evidenced by their
significantly reduced amide proton exchange rates with the
solvent (H2O). Although nearly all polypeptide amide
protons are involved in hydrogen bonds in a globular protein
those in regular secondary structures appear to be longerlived.
• For example, after placing a lyophilized sample of BPTI into
2H O many amide protons are completely replaced with
2
deuterium within 1hr. Over the next several hours, the amide
protons in the N-terminal and then the C-terminal helix also
completely exchange. However, some amide protons
participating in the central antiparallel sheet are still present
after some months.
Selection of Secondary Structural Segment
• Sequential stretches of residues with consistent secondary
structure characteristics (NOEs, coupling constants, slowly
exchanging amide protons, and chemical shifts) provide a reliable
indication of the location of these structural segments. However,
the boundaries of these segments are difficult to define precisely.
Survey of NMR-derived Structural
Parameters Characterizing Reduced Grx3
Shown above, amide proton exchange rates with solvent water (filled
diamonds) kNH < 0.02 min-1, coupling constants: 3JHNa (filled
circles) < 6.0 Hz and (open circles) > 7.0 Hz, and sequential backbone
dNN and daN NOE connectivities are classified as strong, weak, or
absent and are represented by the thickness (or absence) of a bar
connecting the residues in question. Medium range NOE
connectivities daN (i, i+3) and (i, i+4) are drawn as line segments
connecting the residues contributing to the observed cross peak if
present.
NMR-determined Protein Structures
• The multiple-dimensional
NMR spectra used to derive a
number of distance
constraints for different
hydrogen atoms along the
polypeptide chain of the Cterminal domain of a
cellulase. The diagram
shows 10 superimposed
structures that all satisfy the
distance constraints equally
well. These structures are all
quite similar since a large
number of constraints were
experimentally obtained.