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An Introduction to Biological NMR

Spectroscopy*
Dominique Marion‡§¶储
NMR spectroscopy is a powerful tool for biologists inter- physics, chemistry, biology, and medicine. However, it took
ested in the structure, dynamics, and interactions of bio- more than 60 years to reach this interdisciplinary status. The
logical macromolecules. This review aims at presenting in discovery of nuclear magnetic resonance was made inde-
an accessible manner the requirements and limitations of pendently by two groups of prominent scientists, Felix Bloch
this technique. As an introduction, the history of NMR will et al. (1) and Edward Purcell et al. (2) at the end of World War
highlight how the method evolved from physics to chem- II. The 1952 Nobel Prize in Physics was awarded jointly to
istry and finally to biology over several decades. We then
them “for their development of new methods for nuclear mag-
introduce the NMR spectral parameters used in structural
netic precision measurements and discoveries in connection
biology, namely the chemical shift, the J-coupling, nuclear
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Overhauser effects, and residual dipolar couplings. Res- therewith.”
onance assignment, the required step for any further NMR Only a few years after the initial discovery, NMR entered the
study, bears a resemblance to jigsaw puzzle strategy. The field of chemistry when Proctor and Yu (3) accidentally dis-
NMR spectral parameters are then converted into angle covered that the two nitrogens in NH4NO3 gave rise to two
and distances and used as input using restrained molec- different signals. This first observation of the chemical shift
ular dynamics to compute a bundle of structures. When was confirmed one year later by the detection of three lines in
interpreting a NMR-derived structure, the biologist has to the spectrum of ethanol. In 1952 the first commercial Varian
judge its quality on the basis of the statistics provided. NMR spectrometer operating at 30 MHz for 1H was produced.
When the 3D structure is a priori known by other means, In 1953, Overhauser (4) observed that the saturation of elec-
the molecular interaction with a partner can be mapped
trons in metals led to an increase of the nuclear polarization:
by NMR: information on the binding interface as well as on
this effect known later as the “nuclear Overhauser effect” was
kinetic and thermodynamic constants can be gathered.
NMR is suitable to monitor, over a wide range of fre- the first evidence that spins (nuclei or electrons) could com-
quencies, protein fluctuations that play a crucial role in municate through some spin–spin interactions. These double
their biological function. In the last section of this re- resonance methods were also used to detect spin–spin cou-
view, intrinsically disordered proteins, which have es- pling, the other types of interaction between nuclei and in
caped the attention of classical structural biology, are 1961, Freeman and Whiffen (5) analyzed the spin–spin cou-
discussed in the perspective of NMR, one of the rare pling network in 2-furoic acid.
available techniques able to describe structural ensem- In these early years, NMR was a rather insensitive method:
bles. This Tutorial is part of the International Proteomics for instance, pure liquids were required to detect 13C NMR
Tutorial Programme (IPTP 16 MCP). Molecular & Cel- spectra. Stronger electromagnets were designed to reach 100
lular Proteomics 12: 10.1074/mcp.O113.030239, 3006–
MHz for the 1H frequency until the emergence of supercon-
3025, 2013.
ducting magnets in the early 1960s. The first 200 MHz spec-
HISTORICAL PERSPECTIVE trum of ethanol (6) was published in 1964 after solving a great
Nuclear magnetic resonance (NMR)1 is, at the present time, deal of technical challenges such as magnet homogeneity
an established method in a variety of scientific fields such as and stability. A further gain in sensitivity was provided by the
introduction of Fourier transformed (FT) NMR (7) in 1966 by
From the ‡University Grenoble Alpes, Institut de Biologie Struc- Ernst and Anderson, both working at Varian. The ability to
turale (IBS) F-38027 Grenoble, France; §Centre National de la Re- excite simultaneously and then unravel all signals was a meth-
serche Scientifique, Institut de Biologie Structurale, F-38027 odological breakthrough that opens the door to the develop-
Grenoble, France, ¶CEA, DSV, Institut de Biologie Structurale,
F-38027 Grenoble, France
ment of numerous pulse sequences.
Received April 22, 2013, and in revised form, June 18, 2013 In 1957, exploratory studies were undertaken on small bi-
Published, MCP Papers in Press, July 6, 2013, DOI 10.1074/ ological molecules such as common amino-acids and the first
mcp.O113.030239 spectrum of bovine pancreatic ribonuclease (8) was recorded
1
The abbreviations used are: NMR, nuclear magnetic resonance;
1D, 2D, 3D NMR, one-, two-, three-dimensional NMR; CSA, chemical
shift anisotropy; FT, Fourier transform; HSQC, heteronuclear single effect; NOESY, nuclear Overhauser effect correlation spectroscopy;
quantum correlation spectroscopy; IDP, intrinsically disordered pro- PRE, paramagnetic relaxation enhancement; RDC, residual dipolar
tein; ITC, isothermal titration calorimetry; nOe, nuclear Overhauser coupling; r.f, radio-frequency.
3006 Molecular & Cellular Proteomics 12.11

An Introduction to Biological NMR Spectroscopy
at 40 MHz. After failing to observe a spectrum in H2O, these In recent years, biological NMR has evolved toward more
authors reported a 1H spectrum in D2O that exhibited four diverse applications. As depicted in Fig. 2, the number of
lines corresponding to the various types of protons (aromatic published structures solved by NMR has stagnated over the
and aliphatic). Most of the research in the 1960s was carried years in comparison with the structures solved by X-ray dif-
out on synthetic or natural peptides and on some paramag- fraction. This trend can easily be explained by the fact that
netic proteins such as cytochrome c and myoglobin, where solving a protein structure by X-ray can be quite fast once
some resonances fall outside of the standard range of chem- suitable crystals have been obtained. However, NMR can
ical shift. The greatest hurdle was the suppression of the provide other types of information that is hardly amenable by
water signal that is several orders of magnitude larger than the crystallography: dynamics can be investigated by NMR over a
signal of interest. wide range of time scales (12), from slow exchange where the
The next step was the introduction of two-dimensional (2D) two interconverting species are visible to fast motion using
NMR by R. Ernst et al. in 1976 following a clever idea of J. relaxation measurements. In the field of drug discovery (13),
Jeener, a Belgian physicist. The introduction of an additional chemical shift mapping provides information on which part of
frequency axis led to correlation maps (9) between spins the protein is interacting with the ligand and NMR is very
(either via J-coupling or nOe) and to powerful tools for reso- powerful at screening or optimizing hits. In conclusion, the
nance assignment. Today, NMR is very unique in the versa- ecological niche of NMR is currently not restricted to protein
tility of the multidimensional experiments that can be imple- structure determination but covers a wider range of relevant

mented. In 1991, the Nobel Prize in Chemistry was awarded information.
to Richard Ernst “for his contributions to the development of NMR Parameters—A NMR spectrum can only be observed
the methodology of high resolution nuclear magnetic reso- for nuclei that possess a net spin. In this respect, the most
nance (NMR) spectroscopy.” abundant nucleus in a protein, hydrogen, is well suited as its
2D NMR was quickly transferred to the field of biomolecules most abundant isotope (1H) has spin 1⁄2. In contrast, carbon,
by the group of K. Wüthrich. Its feasibility was demonstrated nitrogen, and oxygen are not easily visible by NMR, at least for
on a 10 mM sample of BPTI, a 58 amino acid protein, despite their most abundant isotopes (12C, 14N, and 16O). We will
the then-available limited computational facilities. The very discuss later in this review how to enrich the protein with
high protein concentration, that drastically limited the use of isotopes (“isotope-labeling”) such as 13C and 15N. Although
this new method at that time, has been greatly reduced over these strategies were very expensive two decades ago, uni-
the years as a result of numerous technical improvements. In form or selective labeling is now cost-effective.
1985, the first structure of a small globular protein was pub- NMR experiments are carried out in a static magnetic field
lished (10) but for the well-established community of X-ray B0 (several Tesla) aligned conventionally along the ⫹z axis. As
crystallographers, the reaction was disbelief, claiming that the a result of this field, the space is no longer isotropic and all
obtained structure had been modeled using other previously interactions experienced by the spins will depend on the
crystallized proteins. The credibility of NMR as structural tool orientation of the molecule with respect to the magnetic field
for proteins was strengthened over the years as its perform- B0. In mathematical terms, the anisotropic NMR interactions
ance increased: 3D NMR was introduced first on unlabeled are described by second-rank tensors or 3 ⫻ 3 matrices.
proteins followed quickly by a new set of triple resonance However, in liquid state NMR, the molecule under investiga-
experiments (11) using 15N and 13C labeled samples. In 2002, tion is rotating freely with a correlation time ␶c (1–50 ns) much
The Nobel Prize in Chemistry was awarded to Kurt Wüthrich smaller than the acquisition time: if this rotation is isotropic, all
“for his development of nuclear magnetic resonance spectros- interactions will average out and only the isotropic component
copy for determining the three-dimensional structure of bio- will be observed. This explains the sharpness of resonance
logical macromolecules in solution.” typically seen in solution NMR spectra as compared with
NMR spectrometers devoted to structural biology benefit solid-state spectra.
from several recent technological achievements: (1) higher Chemical Shift—The atomic-resolution power of NMR is
magnetic field (ⱖ 950 MHz) can be reached using new super- intrinsically linked to the occurrence of chemical shift. In a
conducting material, (2) cryoprobes, in which the transmit/ NMR spectrum, the magnitude or intensity of the resonance is
receive coils are maintained at low temperature to reduce the displayed along a single frequency axis (in the case of 1D
noise, have become standard equipment, (3) the design of the NMR) or several axes (for multidimensional NMR). Chemical
spectrometer electronics leads to superb experimental long- shift is usually expressed not in Hz but in ppm relative to a
term stability, and (4) alternate processing methods are pos- standard:
sible with the increased power of computers. Fig. 1 shows a
␯ ⫺ ␯0
recent NMR spectrometer at intermediate field (600 MHz): ␦共 ppm兲 ⫽ 106 · (1)
␯0
most biological studies can be carried out on this midrange
model and could be completed by getting access to a large- where ␯ is the signal frequency in Hz and ␯0 that of a reference
scale facility (⬎ 950 MHz). compound. Thus, chemical shifts in ppm can be compared
Molecular & Cellular Proteomics 12.11 3007


FIG. 1. Picture of a mid-range NMR spectrometer (Courtesy of Bruker-Fällanden). It is composed of a cryomagnet (center), an electronic
console (right) and the user console (left). The strength of the magnet is graded according to the frequency of the 1H NMR signals (here 1H ⫽
600 MHz or B0 ⫽ 14T). The magnet Dewar contains the superconducting coil (not visible) at liquid helium temperature and a detection probe,
which is also cooled at 15–20K to minimize the thermal noise (cryoprobe). The sample is inserted from the top of the bore by means of a
pneumatic lift. The r.f. pulse and pulsed field gradients enter the probe from the bottom of the magnet bore and two insulated pipes provide
the cooling for the cryoprobe and the temperature regulation of the sample (–20 to 80 °C). Most recent magnets are actively shielded, i.e. the
stray field outside the magnet itself is minimized. The electronic console contains a pulse programmer that generates and amplifies r.f. and
pulse field gradient pulses sent to the probe and trigger the data acquisition: modern spectrometers are equipped with four channels (usually
1
H, 13C, 15N, and 2H for the frequency-field lock) with independent frequency synthesis and amplification. Although the list price of NMR
spectrometers has considerably dropped over the years, it does not depend linearly with the magnetic field. The price ratio for 600, 800, and
950 MHz is roughly 1: 2: 6.
between data sets recorded at different field strength. Several ual resonance assignments. If the chemical shifts of com-
calibration standards are available: tetramethylsilane (TMS) is pound A change when compound B is added to the sample, we
used in organic solvents but because of its poor solubility in already know that A and B are interacting. If the resonances of
water, it is replaced by 2,2-dimethyl-2-silapentane-5-sulfonic A have been assigned (see below), then these changes can be
acid (DSS) for protein NMR (IUPAC recommendation). How- interpreted at the atomic level. Through such an experiment
ever, to avoid any additional compound that might interfere applied to a protein-ligand interaction (13), we can learn what
with the protein, most spectroscopists use, as a calibration parts of the small molecule are interacting and to which part of
intermediate, the water line although its position is tempera- the macromolecular target the small molecule is bound.
ture- and pH-dependent. Chemical shift is by essence an anisotropic interaction but
Measuring chemical shift value is the most amenable task we only observe the isotropic part in solution. At high field,
of NMR spectroscopy. The wealth of information provided by chemical shift anisotropy (CSA) can broaden NMR signals for
chemical shift data depends on the availability of the individ- some nuclei (CO in proteins for example) but it can be safely


FIG. 2. Number of structures deposited to the RCSB protein data bank (http://www.rcsb.org/pdb/) over the years. The number of
structures solved by X-ray crystallography is steadily increasing whereas the NMR-based ones have hit a plateau. In the inset, the data for the
early years of crystallography are displayed with an extended vertical scale for clarity. For the former method, the crystallization step remains
a major bottleneck but once suitable diffraction data are available, the structure can be obtained rather quickly. NMR is primarily hampered
by the limitation in protein size that can be studied: despite that resonance assignment and nOe interpretation have been automated, it still
requires more human input during these processes.
disregarded otherwise. The external magnetic field B0 induces

currents in the electronic clouds in the protein; in turn, these
circulating currents generate a local induced field Bind. As a
result, the different spins sense the vector sum of the two
fields:
¡ ¡ ¡
Bloc ⫽ B0 ⫹ Bind
and will thus not resonate at the same frequency. Chemical

shifts are extremely sensitive to steric and electronic effects
and thus in the case of proteins, to secondary and tertiary
structure. Unlike nOe and J-coupling, chemical shift does not
depend on a single pairwise interaction between well-identi- FIG. 3. Without chemical shifts, NMR structural parameters
fied partners: its prediction or quantitative interpretation is could not be measured and interpreted at atomic level resolution.
In fact, the magnetic field at the nucleus is generally different from the
thus more complex. Let us consider the chemical shifts of applied field B0: this additional contribution (or screening) arises from
backbone 15N in proteins: the standard chemical shift range the interaction of the surrounding electrons with the applied field. The
for this nucleus runs from about 100 to 135 ppm, but outliers electron density around each nucleus varies according to its chemical
at 77.1 and 142.81 ppm have been reported. In one of the properties (nature, bonds . . . ): in a protein, the amide protons reso-
largest (723 residues) assigned proteins, Malate Synthase G, nate between 6 and 9 ppm whereas the CH3 groups of aliphatic
residues are between 0 and 2 ppm. In aromatic moieties (as found in
71 alanines have been assigned: 4 Ala 15N exhibit a shift Tyr, Phe, His, and Trp), a ring current is induced by the delocalized
above 130 ppm and 4 below 118 ppm. This clearly shows that ␲-electrons: this current generates a small magnetic field that adds to
a signal cannot be assigned on the basis of the covalent the applied field B0. A proton located in the plane of the aromatic ring
structure of the protein. (Ha) experiences a stronger field whereas another facing the ring (Hb)
As the number of assigned proteins is increasing, greater perceives a weaker field. Ha is said to be downfield shifted whereas
Hb is upfield shifted. These expressions date back to the early days of
insights have been gained into the contribution to chemical continuous wave NMR spectroscopy where spectrometers were op-
shift of torsion angles, aromatic rings (Fig. 3), solvent acces- erating at constant frequency: to observe proton Ha at a constant
sibility, temperature, pH, and ionic strength. Several data- frequency, a weaker applied B0 was necessary.


FIG. 4. Detection of J-coupling and nuclear Overhauser effects in 1D NMR spectra. The reference spectrum (inset A) contains four
signals labeled “1” to “4” in this spectra region. Two signals (“2” and “3”) are singlets and two are multiplets (1 and 4). This multiplicity indicates
the number of J-coupled neighbors but not their assignment. In inset B, the signal of 4 has been continuously irradiated during the recording
of the spectrum (J-decoupling). The 4 signal is completely bleached out and the two lines of the 1 signal collapse: 1 has been decoupled from
4. This experiment indicates that spin 1 and 4 are J-coupled, i.e. covalent neighbors in the molecule. In inset C, the signal of 3 has been
saturated before the recording of the spectrum (1D nOe experiment). 3 disappears and the intensity of 1 is altered whereas the fine structure
remains unchanged. This experiment evidences a dipolar interaction or a nuclear Overhauser effect between C and A: these two nuclei are
spatial neighbors (⬍ 5–7 Å): the absence of J-coupling between them indicates that they are separated by more than five chemical bonds. The
same effect could be detected if spins 1 and 3 would belong to two different but interacting molecules.
bases are available over the internet as chemical shift repos- nances. This observation is the basis of the Chemical Shift
itories: the largest one is the BioMag-ResBank (http:// Index (CSI) (18, 19), a method that uses chemical shifts to
www.bmrb.wisc.edu), which contains 7800 entries (as of identify the type and location of protein secondary structures
2012). Smaller curated databases, where the data found in the along a protein chain. As compared with circular dichroism
BioMag-ResBank have been selected and corrected, have (CD) spectra that are used to determine the global protein
also been generated such as TALOS or TALOS⫹ (14) for more secondary structure content, the CSI method provides infor-
specific purposes. mation at the residue level. Without resource to nOe meas-
For each type of amino acid, chemical shifts can be inter- urements (see below) and structure computation, the second-
preted in terms of secondary structure by subtracting refer- ary structure of proteins can be obtained from chemical shifts.
ence values for random coil structures. Data obtained in the Along the same lines, the chemical shifts can also be used
1970s on 1H shifts on small peptides Gly-Gly-Xaa-Ala (15) to directly derive torsion angles. The backbone conformation
have been recently supplemented by 13C and 15N data in is defined by two dihedral angles (␾ and ␺) for each amino
various aqueous and organic solvent conditions (16) and are acid as well as several angles for the side-chain (␹1, ␹2. . .).
available at the BioMag-ResBank. These random coil values TALOS uses a database of protein sequences, chemical shifts
can be further improved by integrating nearest-neighbor and dihedral angles to predict backbone dihedral angles, but
effects. fails to make any prediction only for roughly 30% of the
Beside random coil values, reference values for ␣-helices residues. The success of the TALOS (20) methods (and its
and ␤-sheet (17) have been assembled from NMR data for improved version TALOS⫹) is clearly illustrated by the high
each residue type in experimentally observed secondary number of citations of the original paper (⬎ 2000 citations).
structure. For the 15N shift in Ala, a reference value of 121.4 Ongoing research is currently aimed at computing protein
ppm is found in ␣-helices, 124.5 in ␤-sheet and 123.6 in structures using only chemical shift information: the goal of
random coils. As far as carbons are concerned, the C␣ and this strategy, which can immediately follow the resonance
CO move to higher chemical shifts in ␣-helices and to lower assignment, is to evade the lengthy process of nOe assign-
shifts in ␤-strands but the trend is reversed for the C␤ reso- ment (see below). This approach makes use of the Rosetta

TABLE I than larger proteins. As a result, the detection of 4JHH and

Typical values for the scalar coupling in proteins 5
JHH couplings, which is straightforward in peptides, be-
2 1
JHH 9–15 Hz JNCO 15 Hz comes unrealistic on a 20 kDa protein. By comparing the
3
JHH 0–14 Hz 1
JC␣CO 55 Hz
magnitude of the 1H-1H coupling 2JHH and 3JHH with that of
1
JNH 90 Hz 1
JC ␣C ␤ 35 Hz
1
JNC␣ 7–11 Hz 1
JCH (aliphatic) 130–150 Hz the heteronuclear one (1JNH, 1JCH . . .) (see Table I), one
2
JNC␣ 4–9 Hz 1
JCH (aromatic) 160 Hz readily understands why the 1H-based strategies used in the
70’s for protein resonance assignment have been superseded
by the triple-resonance approach (see below) relying on much
algorithm for de novo protein modeling. This algorithm builds larger heteronuclear couplings.
a large number of models for the protein on the basis of As scalar couplings stem from the bond orbitals, they all
fragments from the PDB database that share some sequence contain structural information. One-bond couplings show little
similarity: only the models that are compact and energetically variation for a type of spin pairs: however, 1JCH for aliphatic
favorable are retained. In the CS-Rosetta approach (21), carbons (sp3) are smaller than for aromatic carbons (sp2) and
backbone chemical shifts are used to select suitable frag- for each hybridization, a rough correlation with the 13C chem-
ments: with this additional information, the convergence of ical shift has been reported (23). Bax and coworkers (24) have
this Monte-Carlo algorithm requires a smaller number of mod- analyzed the variation of the 1JC␣H␣ in proteins [135 Hz -150
els and thus smaller amounts of computing time, at least for Hz] and reported a empirical correlation with the backbone

proteins of relatively simple topology. dihedral angles (␾ and ␺) of the residue. With this limited
Scalar Coupling—Scalar coupling (or J-coupling) is a variation of the 1J, heteronuclear correlation experiments
through-bond interaction between two nuclei (A and X) with a (such as HSQC or HMQC) could be designed to yield 1H-X
nonzero spin. It is an indirect interaction between the two cross-peaks of homogeneous amplitude.
spins that is mediated by the electrons: one spin perturbs the Similarly, 2J couplings (2JC␣N, 2JHNC␣ . . . ) show empirical
spins of the shared electrons, which in turn will perturb the correlations with ␾ and ␺ angle but the difficulty lies in the
second spin. Only the isotropic part of the anisotropic inter- interpretation of the simultaneous dependences on more than
action is detected in liquid-state NMR. Reported in Hz, it is a single torsion angle (25). By far, the most valuable structural
field-independent and causes NMR signals to be split in information is derived from three-bond mediated vicinal cou-
multiple peaks: if two spins 1⁄2 are scalar coupled, the plings (3J): in the early 1960s Martin Karplus (26) established
spectrum of each will be a doublet (see Fig. 4) and the a relationship between the dihedral (torsion) angle (⌽) be-
separation between the two lines is the coupling constant tween protons (H-C–C-H) and vicinal coupling 3J. The general
JAX. The presence of two lines can be understood as two form of the Karplus relationship is:
distinct populations of spin A: the spins A, which have a
neighbor X in the “up” spin state (1) (i.e. aligned along the 3
J共⌽兲 ⫽ A cos2共⌽兲 ⫹ B cos(⌽) ⫹ C (2)
magnetic field ⫹z), will resonate at ␦ ⫹ 1⁄2JAX whereas the
spins A, which have a neighbor X in the “down” spin state and the coefficients A, B, and C are parameterized for each
(2), resonate at ␦ - 1⁄2JAX. combination of nuclei.
The indirect interaction may either increase or decrease the In proteins, the 3JHNH␣ coupling provides information on
resonance frequency: the absolute sign of a J-coupling can- the ␾ backbone angle whereas the 3JH␣H␤ provides informa-
not be experimentally determined by NMR, but only the relation on the side-chain ␹1 angle (21). In ␣-helices (␾ ⫽ ⫺64° ⫾
tive sign of two couplings sharing a common nucleus. Scalar 7°), a small 3JHNH␣ is observed (⬍ 4 Hz) whereas for ␤-sheets
couplings are denoted as nJAX, in which A and X are the (␾ ⫽ ⫺120° ⫾ 10°) larger couplings are present (⬎ 4 Hz).
interacting nuclei and n the number of covalent interceding Unfortunately, no 3JHH coupling is linked to the other back-
bonds. One-bond coupling (1J) are an order of magnitude bone angle ␺, which can be obtained in a 15N labeled peptide
larger than two- and three-bond couplings (2J, 3J), which in using the much smaller 3JH␣N.
turn are larger than long-range coupling such as 4J and 5J The ␤-methylene moiety found in most amino-acids is a
(22). Typical values for couplings observable in proteins are prochiral center, i.e. it could become a chiral center by re-
reported in Table I. placing one of the two protons by another group (a deuterium
For experimental purposes, the magnitude of any scalar for instance). As a result, the pro-R and the pro-S protons
coupling should always be compared with the line-width (⌬␯) have different chemical shifts. Their stereospecific assign-
of the associated signals. A coupling smaller than the line- ment is achieved by combining several vicinal coupling con-
width is hardly visible on the 1D NMR spectrum and a 2D stants (3JH␣H␤1, 3JH␣H␤2, 3JNH␤1, 3JC’H␤1 . . . ) and several
correlation experiment through this coupling will have a low distance measurements based on nOe information (see be-
efficiency and thus poor sensitivity. As discussed below, the low). Similarly, the two CH3 in Leu and Val isopropyl groups
NMR line width increases with the size and rigidity of the need to be stereospecifically assigned. It has been shown that
molecule and small peptides exhibit much narrower signals the availability of stereospecific assignment for these prochi-

ral centers improves the accuracy and the precision of the The nuclear Overhauser effect (nOe) (4) was introduced in the
derived NMR structures (27). historical section as the intensity variation of a metal ion spec-
How could a scalar coupling be evidenced in a NMR spectrum when their electrons were irradiated. In peptides or pro-
trum? In crowded spectral regions a doublet could be mis- teins, nOe refers to intensity alteration of a spin resonance when
taken for two independent resonances. Decoupling methods other nuclei are irradiated (30). An example is given in Fig. 4: in
have been used since the early days of NMR spectroscopy the lower spectrum, the resonance of spin 3 is saturated leading
and are illustrated in Fig. 4. In this figure, the spin 1 exhibits a to an increase of the amplitude of spin 1 as compared with the
doublet as a result of a scalar coupling with spin 4. If one reference spectrum. This effect, also called cross-relaxation, is
continuously irradiates spin 4 while recording the spectrum the evidence of a dipolar interaction between spins 1 and 3.
(this is called “decoupling”), the two lines of 1 will collapse. In Note that in contrast with the J-decoupling experiment, the
presence of decoupling, one can no longer consider two multiplicity of spin a is not altered. As electrons are not medi-
distinct populations of 1 spins (the one next to 11 and the ating the interaction as for J-coupling, nOe is a short range
other next to 12): their fast interconversion between the two through-space interaction (there is an 1/rAX6 dependence on
lead to an average resonance frequency for “a.” Nowadays, distance). This property is pivotal for the application of NMR to
these double-irradiation techniques are no longer used and structural biology (31): interstrand nOe can be detected in
the proof that two spins are scalar coupled can be obtained ␤-sheets, providing not only the nature of the ␤-sheet (parallel or
antiparallel) but also the register of the strands.
more conveniently from 2D correlation experiments (COSY or

The nOe dependence on dynamics is depicted in Fig. 5 (see
DQF-COSY (28)).
caption for details). In a protein with nonuniform flexibility,
Nuclear Overhauser Effect—The second kind of anisotropic
nOe can only be qualitatively converted into distances. An-
spin interaction is the dipolar coupling. Though much larger
other annoyance is the phenomenon known as spin diffusion,
than the scalar coupling (DNH ⬎ 22 kHz), it is not directly
i.e. the spreading of the nOe along a chain of nuclei. In fact,
visible in liquid-state NMR spectra. All we have to keep in the magnetization can move to another nucleus by cross-
mind at this stage is the fact that the dipolar interaction relaxation with a much faster and efficient rate than it decays
between A and X depends on the internuclear distance (rAX) by auto-relaxation. An analogy in thermodynamics is the heat
¡
and on the orientation of the vector AX with respect to the transfer from an object to a chain of neighbors which occurs
magnetic field. much faster than the thermal dissipation. After numerous
Because of the isotropic tumbling of the molecules in so- studies in the 1980s to investigate the pitfalls in the conver-
lution, the dipolar coupling averages to zero during the time sion of nOe into distances, the following strategies have been
needed to record the spectrum. It can be detected either in established: the identification of a large number of qualitative
solid-state NMR or when the environment in liquid is no longer nOes should be preferred to a small set of accurate distances.
isotropic. This later circumstance is encountered when lipid Since the introduction of two-dimensional NMR in the
bicelles or bacteriophages are introduced in the sample to 1980s, nOe correlations are no longer obtained by double-
induce partial alignment (29) (see section on residual dipolar irradiation methods (one spin is irradiated and the others are
couplings). monitored). NOESY experiments (32) provide the same type
So far we assume that the molecule tumble isotropically in of information in a more compact manner. It is of interest to
solution. The dipolar interaction acts on the spin system via notice that the NOESY pulse sequence was designed to inves-
relaxation mechanisms. Relaxation is the process by which tigate any type of exchange process by NMR: a chemical ex-
nuclei regain their thermal equilibrium after being perturbed change of a species A interconverting into a species B or
magnetization transfer between two spins in the same molecule
by radiofrequencies. Without relaxation, no NMR experiment
(A 7 X). The two-dimensional approach is handier to use be-
could ever be repeated! NMR experiments are carried out in
cause all neighbors are identified in a single experiment: how-
solution where proteins undergo two types of motion as a
ever, the spin diffusion issue mentioned earlier remains a severe
result of collisions with other proteins and solvent molecules:
penalty for deriving accurate distances from NOESY spectra
an erratic random global movement, called Brownian motion,
(33).
and internal fluctuations at the level of residues, secondary
Residual Dipolar Coupling—In the previous section, we
structure elements or domains. All these motions modulate
¡ have reported that, in solution, the dipolar coupling is gener-
the orientation of the AX vector with respect to the magnetic ally averaged to zero by isotropic motion (see Fig. 6a) and can
field and thus the AX dipolar interaction. This modulation only be indirectly detected through relaxation effects. In 1995,
generates radiofrequency (r.f.) fluctuations that contribute to Tolman et al. (34) showed that some small residual dipolar
bring the magnetization back to its equilibrium. The spins coupling could be observed in cyanometmyoglobin because
have been coherently moved away from equilibrium by r.f. of the presence of a paramagnetic ion in this protein. The
pulses and return to this state incoherently by means of anisotropic magnetic susceptibility of this protein gives rise to
motion-induced r.f. fluctuations. a weak alignment and residual dipolar couplings (RDC) were


FIG. 5. Nuclear Overhauser effect in a homonuclear (1H) two-spin system (A and X). The four energy levels (11, 12, 21, 22) are
populated according to a Boltzman distribution (inset A). In the unperturbed case, the population difference is the same for all transitions (⌬ ⫽
4). If the A-spin transitions are saturated, the associated populations are equalized (inset B). Then the cross-relaxation mechanisms will attempt
to re-equilibrate the populations toward the equilibrium shown in inset A. The way this re-equilibration will occur, depends on the frequencies
generated by the molecular fluctuations: If they are fast (as in a small peptide), the double-quantum transition W2 (between 11 and 22) at
␻A⫹␻X will be most efficient (inset C). In contrast, a large protein will emit mainly low frequency fluctuations, which correspond to the zero-quantum
transition W0 (21 to 12) at ␻A–␻X (inset D). The outcome of the re-equilibration process will thus be opposite: an intensity increase of the X-spin
transition (⌬ ⫽ 6, positive nOe) is observed for small molecules and a decrease (⌬ ⫽ 2, negative nOe) for the larger proteins.
proportional to the square of the static field. Bax and cowork- interactions often can be measured: large 1DCH and 1DNH but
ers (35) were able to reproduce similar results on a diamag- also smaller 1DCC and 1DCN couplings.
netic protein but the observed effects were too weak to have What kind of structural information is provided by the RDC?
any practical use (1DHN ⬍ 0.2Hz). For ease of understanding, we first assume that we know the
Various options for increasing the degree of molecular preferential orientation of the protein in the alignment media.
alignment have been searched for. Proteins are generally This orientation (or alignment tensor in a mathematical for-
nonspherically symmetric molecules and when dissolved in a malism) can be visualized as an ellipsoid (Fig. 6C) mapped on
solvent containing molecules that are oriented relative to the the molecular frame (Fig. 6D). The experimental RDC are give
magnetic field, a degree of alignment is transferred to the as function of the characteristics of the ellipsoid along the
protein (Fig. 6B). Mixtures of DHPC (hexanoyl-phosphatidyl- three dimensions: Azz (the main direction) Axx and Ayy):
choline) and DMPC (dimyristoyl phosphatidylcholine) with
molar ratio between 1:2 and 1:35 form disclike assemblies in
solution that align in the magnetic field. In this type of media,
Dij共␪,␾兲 ⫽ ⫺ K 冋冉
␥i␥j
rij3
Azz 3 cos2 ␪ ⫺ 1 冊
1
H-15N RDC (1DHN) ranging for ⫺10 to ⫹10 Hz could be
measured for ubiquitin (28), a small protein that deviates only
weakly from isotropic diffusion.
冉冋
⫹ Axx ⫺ Ayy sin 2␪ cos 2␾ 册 (3)
Over recent years, a number of alignment media compatible This equation can be simplified when the tensor or the
with proteins have been proposed: oriented bilayers, filamen- ellipsoid is axially symmetric (Axx ⫽ Ayy) as:
tous phages, rod-shaped cellulose particles, purple mem-
冋册
brane fragments, lyotropic alcohol-based mixtures (36), and ␥i␥j
mechanically stressed gels (37). The medium should be stable Dij共␪,␾兲 ⫽ ⫺ K 3 Azz共3 cos2 ␪ ⫺ 1兲 (4)
rij
over several days at the temperature and pH suitable for the
NMR study and the protein should remain soluble and possi- Although the two above equations seem at first glance
bly monomeric. To ascertain that the protein structure is not complex, the main merit of RDC measurements can be un-
altered by the presence of the alignment medium, chemical derstood from Fig. 6D: Each measured RDC provides orien-
shifts can be used as a probe. For relatively well-behaved tational information with respect to a global frame of refer-
systems (less than 20 kDa), many different types of dipolar ence. Although RDC and J-coupling both provide angular

Despite the richness of information contained in RDC, sev-

eral bottlenecks should be mentioned for their measurement
and interpretation. Finding suitable alignment media for a
given protein may require numerous attempts. Most new me-
dia have been first described on test proteins such as ubiq-
uitin, known to be highly well-behaved and stable over long
periods of time. The accurate measurement of numerous RDC
is a time-consuming task requiring spectrometer time and man-
ual interpretation. The alignment tensor has to be deduced and
oriented with respect to the protein using global fitting of all
measured RDC in one media: once this orientation is known, a
large number of potential solutions for the orientation of each
inter-dipolar vector correspond to each measured RDC value.
The orientational degeneracy continuum for a single RDC can
be lifted by measuring multiple couplings and by using several
media leading to differing alignment properties (40).
Two-dimensional NMR and Beyond—The wealth of infor-

mation that can be obtained by NMR relies on multidimen-
sional NMR. Any structural investigation starts with the re-
cording of a standard one-dimensional NMR spectrum. This
spectrum bears resemblance with spectra obtained with any
optical spectroscopy (infrared, visible, ultraviolet): absorption
is plotted as function of a frequency or wave-length. For each
nucleus, the NMR spectrum displays a signal at a given reso-
nance frequency. We have described earlier that the absolute
FIG. 6. In an isotropic medium (such as an aqueous buffer), a
diamagnetic protein tumbles nearly isotropically, i. e. all orienta- frequency scale is more conveniently replaced by a scale in
tions have the same probability (inset A). As a result, the dipolar ppm to permit comparison between spectra recorded at differ-
interaction between two spins goes to zero and is only visible through ent fields. In practice, the 1D NMR spectrum is not recorded by
relaxation parameters. When a weak alignment medium is introduced sweeping through the entire frequency spectrum: spins are
(inset B), the protein acquires a small preferential orientation: the
collectively excited by a strong radiofrequency (r.f.) pulse and
incomplete averaging of the dipolar interaction leads to residual di-
polar couplings (RDC). Between the protein and the alignment me- the resulting signal is then sampled. Its Fourier transform leads
dium, the interaction is generally steric but in nonneutral media, to the standard spectrum, i.e. absorption versus frequency. An
long-range electrostatic interactions are dominant. The preferential enormous gain in sensitivity is afforded by this method.
orientational averaging of the molecule can be described by an align- In the early 1970s, two-dimensional NMR (9, 31) was intro-
ment tensor with eigenvalues Axx, Ayy and Azz (inset C) in the molec-
duced following a visionary lecture by Jean Jeener: it has
ular frame. The measured RDC between two spins depends on the
orientation of the internuclear vector with respect to the alignment been widely used since to correlate the resonance frequen-
tensor (inset D) described by the polar coordinates (␾ and ␪). Note cies of several nuclei. In contrast to optical spectroscopy, the
that several degenerate orientations are compatible with data re- information content of a NMR frequency is rather low whereas
corded in a single alignment medium, a degeneracy that can be lifted a correlation experiment mediated by an interaction (J-cou-
using media with different steric and electrostatic properties.
pling or nOe) provides the nature of the partners as well as the
information, it is important to stress a key difference. A vicinal interaction strength.
J-coupling provides relative information, i.e. the orientation of A two-dimensional NMR experiment can be sketched as:
one bond with respect to another one (cf. Eq. 3). A RDC
supplies global information, i.e. the orientation of a bond with
respect to a global frame. When only J-couplings are used for
computing a protein structure, the experimental errors accu-
mulate, leading to distorted conformations. RDCs provide
appropriate remedies primarily for elongated molecules (such
as highly asymmetrical RNA or DNA) or for multidomain pro- During the preparation, the spins are allowed to recover
teins. RDC provide fast answers to specific structural ques- from the previous experiment, they evolve then during a vari-
tions such as conformation changes because of local muta- able evolution delay (t1). The key step in a 2D experiment is
tion or ligand binding. Similarly RDC can determine very the mixing, which allows the magnetizations (or the coher-
accurately the relative orientation of domains of known struc- ences in NMR jargon) to exchange (A ^ B) through any
ture or that of interacting partners (38, 39). interaction (J-coupling or nOe). Finally, the signal is sampled

FIG. 7. 1H-15N heteronuclear single

quantum correlation spectrum (HSQC)
of B. subtilis L,D-transpeptidase LdtBs
(169 residues, concentration 0.7 mM],
pH 6.5) recorded at 600 MHz (43). The
horizontal axis corresponds to the 1H
frequency and the vertical one to the 15N
frequency. A correlation peak is ob-
served for each backbone 1H-15N pair
(all residues except the N terminus and
Pro) as well as for the side-chain NH2 in
Asn and Gln residues. For the side-
chains (see 15n ⫽ [110, 114 ppm], 1H ⫽
[6.5, 8.0 ppm]) two cross-peaks at the
same 15N frequency are detected as the
two 1Hs are not magnetically equivalent.
Two 1D slices taken at the position of the

dotted arrows are shown: in the vertical
slice are visible all amide 15N that bear a
1
H at 7.5 ppm. This illustrates how the
introduction of an additional dimension
(15N) resolves spectral overlaps in the
other one (1H). Note that side-chain 15N
resonances can also be observed for
Arg, His, and Trp.
during the detection period (t2). Although two frequency la- vertical taken through the 2D spectrum— bear resemblance
beling periods are present, the signal is indirectly detected with 1D NMR spectra, but with a reduced number of peaks
during t1, because of the “memory” of the spins. As a matter and thus of overlaps. The large chemical shift dispersion in the
1
of fact, as long as the delays are not longer than the corre- H dimension provides evidence of a well-folded globular
sponding relaxation times, the spins remember their previous protein: if the protein is partially disordered, the correspond-
evolution: the signal detected at the very end of the pulse ing 1H resonances will cluster between 8 and 8.5 ppm (45).
sequence (during t2) is modulated either in amplitude or in Such an HSQC spectrum is nowadays often the very first
phase as a function of t1. The resulting data set will be a (n ⫻ NMR spectrum recorded on a new protein under investiga-
m) matrix of points, corresponding to n time increments along tion: the chemical shift dispersion is a reliable proof of the
t1 and m increments along t2. After applying a 2D Fourier compactness of the protein whereas the line-width of each
transform to the time domain data, a two-dimensional NMR signal provides information on the aggregation state of the
spectrum is obtained. protein (cf (46). for more examples). An HSQC is a very robust
This generic 2D NMR scheme can be used to generate a experiment that requires only a on a small amount of 15N-
so-called homonuclear spectrum (it correlates 1H with 1H labeled material (less than 2 mg for a 20 kDa protein) and only
frequencies) or more generally heteronuclear ones (1H - 15N or half an hour of spectrometer time.
1
H - 13C) (see (46) for more details). Though a number of The modular design of 2D NMR can be easily extended to
variants have been conceived from each basic type, pulse 3D and even 4D NMR. A 3D NMR experiment is described as:
sequences are identified by their acronym: COSY (9) or
Preparation ⫺ Evolution (t1) ⫺ Mixing#1 ⫺ Evolution (t2)
TOCSY (41) for J-coupling 1H — 1H correlation, NOESY (42)
for nOe 1H — 1H correlation, HSQC (43) for 1H - X correlation ⫺ Mixing#2 ⫺ Detection 共t3)
via the JHX coupling. Fig. 7 shows a 1H-15N HSQC spectrum
recorded on a 15N-labeled transpeptidase (169 residues) (44): The resulting spectrum is a three-dimensional spectrum,
the horizontal axis refers to 1H chemical shifts and the vertical with three frequency axes (F1, F2, and F3), which correlates
to 15N shifts. A correlation peak is visible for each pair of three different nuclei (11). A 3D pulse sequence can be envi-
1
H-15N nuclei in the protein as well for some side-chains (Asn sioned as a chemical synthesis with two steps (the mixing
and Gln). The two cross-sections— one horizontal and one building blocks): the nature of the reactants, intermediate and

FIG. 8. The jigsaw puzzle analogy for NMR resonance assignment. Within such a puzzle, the global placement of any individual piece
cannot be inferred just by its shape and the picture depicted on it. However, its position relative to other pieces can be determined by evaluating
the match of the edge profile and the picture. As several candidates can be a priori considered, they are ranked according to a penalty function
that accounts for the complementary match of protuberances and pictures. The manufacturing of the pieces is imperfect, leading to some
looseness and thus a threshold is defined for the penalty function. In panel (A), two pieces have been already successfully matched. Two
possible candidates as neighbors on the right hand side are shown in panel (B): although their shape on the left hand side fits roughly the profile
of the already matched pair, only one of the two could be anchored effortlessly in panel (C). This conservative strategy is essential to complete
the puzzle because any piece that is forced at an incorrect place will be missing somewhere else. The choice made in panel (C) is confirmed
in panel (D) as the edge of the puzzle is reached. Once the complete puzzle is solved (panel (E)), it becomes evident that the piece that was

not chosen in panel (C) fits somewhere else. The time required to complete a jigsaw puzzle depends on three factors: the number of pieces,
their dissimilitude and the looseness of the match. Similarly, an NMR assignment will be more difficult for a larger protein with moderate
chemical shift dispersion if the spectra exhibit poor spectral and digital resolution.
final products is identified during the periods t1, t2, and t3 crystallography, does this ease the resonance assignment of
respectively. As for chemical reactions, the overall sensitivity my protein? The answer is unfortunately negative. Numerous
of a 3D NMR relies on the efficiency of the individual transfers effects control the NMR chemical shifts and thus, even for a
and the most sensitive experiments uses exclusively large 1J protein with a known structure, it is nearly impossible to
couplings (cf. Table I). This observation has led to the design predict them. In other terms, the only way to assign reso-
of the triple resonance experiments that will be discussed in nances is experimental by means of suitable correlation ex-
the next section. periments. For lack of being able to directly link a resonance
NMR Resonance Assignment—NMR resonance assign- to a nucleus, one will attempt to connect each signal with
ment is a prerequisite for studies where one aims at deriving another, with the ultimate goal of revealing a resonance net-
information at the atomic level. Although changes in the spec- work with the same topology as the spin network. Accidental
trum can be monitored even without assignment, the wealth resonance overlaps make assignment more challenging: sig-
of information is greatly enhanced for assigned signals. Let us nal discrimination is limited by the spectral resolution (i.e. the
consider a titration experiment where an unlabeled protein B linewidth of each signal) and the digital resolution (i.e. the
is added to a 15N-labeled protein A: if the spectrum of B varies number of experimental points per Hz). The process of NMR
as a function of the concentration of A (some signal shifts or resonance assignment can be best understood using the
widens), one can already conclude that A and B interact. Note jigsaw puzzle analogy illustrated in Fig. 8. In a puzzle, one
that several biophysical methods (such as fluorescence, aims at finding the position of each piece with respect to its
fluorescence resonance energy transfer, surface plasmon res- neighbors whereas in resonance assignment one wants to
onance etc.) may provide the same information at a much correlate the resonance of a spin with those of the adjacent
lower cost than NMR. nuclei.
We mentioned earlier the unique value of NMR, i.e. distinct Fig. 9 depicts the two procedures that have been conceived
signals can be resolved even for chemically identical groups over the years for resonance assignment:
that are located in different environments in a protein. Con- The original one based on two-dimensional 1H-1H NMR
sequently, for well-resolved spectra (narrow lines and optimal (proposed in the early 1980s),
digital resolution), one expects to discern one signal for each The second one on 3D triple resonance (1H- 15N- 13C) NMR
active spin (1H, 13C, or 15N). Before any data can be obtained (designed in the 1990s).
from spectral parameters, the resonances should be as- Both procedures capitalize on the linear copolymer nature
signed, i.e. a one-to-one correspondence between a nucleus of proteins by correlating resonances belonging to residue (i)
in the molecule and a resonance in the spectrum should be and (i⫹1). The former uses the J-coupling and the nOe,
established. whereas the later relies exclusively on the J-coupling. When
Biologists, who intend to collaborate with an NMR spec- the protein spectra get assigned, the 3D fold of the protein is
troscopist, frequently raise the following question: the struc- not yet known and distance based correlation experiments
ture of a homologous protein has been resolved by X-ray are more problematic than correlations via J-coupling, i.e.

or TOCSY (40) based on J-couplings (see above) and an

inter-residue correlation (NOESY (41)) using nOes. The
NOESY experiment is makeshift in the absence of JHH-cou-
pling through the peptidic linkage. As a result of the protein
fold, two protons can be close in space without belonging to
an adjacent residue; thus, NOESY experiments are optimized
to detect only short distances at the expenses of sensitivity.
The homonuclear strategy has been successful on a number
of small well-behaved proteins (less than 80 –100 residues).
As the molecular weight of the protein increases, its adequacy
weakens for two reasons: (1) the linewidth increases strongly
degrading the efficiency of the J-based correlation and (2)
accidental overlaps generate ambiguities in the puzzle. The
resonance set depicted in red in Fig. 9 illustrates how partial
overlap makes the assignment more intricate. When several
puzzle pieces (cf. Fig. 8B) have similar shapes, their match
with other pieces has to be examined more carefully to avoid

incorrect matches if two pieces would be forced together.
Why did the heteronuclear 3D strategy emerge in the early
1990s? Using a 15N- 13C labeled protein gives the opportunity
of conceiving correlation experiments exclusively based on
J-coupling and some of the heteronuclear couplings are sub-
stantially larger than JHH (cf. Table I). A transfer via a large
J-coupling remains efficient even if the signals are broad.
Switching from 2D to 3D NMR also permits one to cope with
heavier proteins because accidental resonance overlaps are
less frequent.
Triple resonance experiments establish connectivities be-
tween adjacent residues using 1J and 2J couplings: experi-
ments are always used in pairs (HNCO and HN(CA)CO, HNCA
and HN(CO)CA . . . ) to connect residue (i) with residue (i⫹1).
FIG. 9. Strategies for sequential resonance assignment in pro-
teins. As proteins are linear copolymers, these strategies aim at The acronyms used refer to the correlated nuclei and a nu-
correlating the resonance frequencies of one residue with those of the cleus denoted with parentheses is used as a relay but not
following one. 2D-1H-1H correlation experiments can be employed on identified (see (47, 48) for a review). Fig. 9 features the com-
unlabeled proteins and 3D triple resonance experiments are better bined use of HN(CO)CA and HNCA (49) experiments. In the
suited for 15N-13C uniformly labeled molecules. The first approach
optimal case, one can thus track the entire polypeptide chain
uses two correlations experiments: one using 1H-1H J-couplings
(COSY or TOCSY) and one using 1H-1H nOe (NOESY). One has to (with the exception of Pro), but supplementary evidence from
resort to nOe to link two adjacent residues as no 3JHH coupling is other experiment pairs is required in practice for heavy peak
available for this purpose: note that nOe is a through-space effect and overlaps. The intrinsic sensitivity of these triple resonance
that nuclei close in space but not belonging to an adjacent residue experiments depends on the nature of the correlated spins
may lead to fallacious correlations. This issue is resolved in the 3D
(and the coherence pathways between them) and also on the
triple resonance strategy, which relies exclusively on J-couplings.
Triple resonance experiments always works in pairs as illustrated resonance line-width. Thus, it is always difficult to anticipate
here: HN(CO)CA that correlates the HN and the N of residue (i⫹1) with how laborious a resonance assignment will be: molecular
the C␣ of residue (i) and HNCA that correlates the HN and the N of aggregation or internal flexibility will broaden locally the res-
residue (i⫹1) with both the C␣ of residue (i) and that of residue (i⫹1). onances and lead to missing or weak correlation peaks.
In the HN(CO)CA, the carbonyl 13C act as a relay but its frequency is
With this set of triple resonance experiments (47), the back-
not detected. The experimental combination links each (HN, N) pairs
with the preceding and following C␣’s and reciprocally each C␣’s with bone resonance (usually up to the C␤) can be assigned.
the adjacent (HN, N) pairs. Extending the assignment to the entire side-chain is a more
tedious and time-consuming task. Do we need to completely
along the covalent structure. This is one of the rationales why assign the side-chains? Yes, if one wants to determine the
larger molecules are nowadays assigned exclusively using 3D complete 3D structure of the protein. In a number of cases,
triple resonance NMR. where the X-ray structure is already available, a NMR study is
For the 1H-1H approach (cf. Fig. 9), two types of experi- initiated not to confirm the conformation but to answer ques-
ments will be employed: an intraresidue correlation (COSY (9) tions that could not be addressed by other means. With only

the backbone assignment, valuable information can already

be obtained: the location of the secondary structure elements
(␣-helices and ␤-sheets), the flexibility of the backbone over
several time scales (ns, ms . . . ), the affinity and binding site of
a ligand. To identify the side-chain resonance, a combination
of several 3D experiments are employed, some based on
J-coupling transfer (HCCH-TOCSY experiments (50)), some
based on nOe effects (13C edited NOESY). Because spectral
overlap is more severe for backbone nuclei, side-chains are
generally less completely assigned, an issue that can impact
on the precision of the derived structures. The stereospecific
assignment of the prochiral centers complicates even more
the issue: in most amino-acids (with the exception of glycine)
the ␣-carbon is a chiral atom and thus the ␤-carbon is a
prochiral center. As a result of the steric hindrance, the two H␤
exhibit different chemical shifts, as do the two CH3 groups in
valine and leucine. Their stereospecific assignment is gener-

ally obtained by combining J-coupling values and nOe dis-
tances (51), but conformation and dynamics of the side-
chains may prevent gathering this information.
Molecular Interactions by NMR—Protein–protein interac-
tions play a key role in numerous cellular processes. Even
when two partners have been structurally characterized, co-
crystallization of the complex may be difficult because of the FIG. 10. NMR detection of molecular interactions. A protein (P) is
low affinity or some local disorder. NMR can complement interacting with a ligand (L) (another protein, a small molecule, a RNA
fragment . . . ) with an association constant Ka (inset A). This thermo-
these studies primarily for weak interactions (Kd ⬎ 100 ␮M).
dynamic constant is related to two kinetic constants, the on-rate
Fig. 10 summarizes various NMR tools for complex studies: constant kon and the off-rate constant koff. Ka ⫽ kon/koff ⫽ [PL]/[P].[L].
chemical shift perturbation, paramagnetic relaxation en- The affinity between the two molecules and structural information on
hancement, intermolecular nOe, H/D exchange rates and re- the complex can be obtained using several NMR parameters. An
sidual dipolar couplings (52). A prerequisite is the resonance unlabeled binding partner is added to a 15N labeled protein: this
causes changes in the environment of the protein and therefore the
assignment of one of the partners, at least for the backbone.
chemical shifts of nuclei at the binding interface (inset B). A paramag-
The proteins are expressed and purified separately and, by netic tag is added to the ligand (inset C): resonances in the protein
selective isotopic labeling methods (13C versus 12C or 15N that are close to the tag are broadened (or even disappear) while
versus 14N), the spectrum of either partner can be hidden. others remains unaffected. Paramagnetic relaxation enhancement
Widely used, the chemical shift perturbation (CSP) method (PRE) provides structural restraints up to 30 Å. Short internuclear
distances can be detected between nuclei belonging to both partners
has been introduced in the early 90’s (53, 54) and is based on
using intermolecular nOes (inset D): discrimination between intra- and
the observation of the spectrum of one molecule (a 15N-1H intermolecular nOe can be simplified by specific isotope labeling of a
HSQC spectrum for instance) with increasing concentration of single partner. Residual dipolar couplings provide information of the
the partner. When the complex is formed, the two molecules orientation of internuclear vector with respect to the molecular frame
are in equilibrium between their free and bound states. This of alignment. If the protein and its ligand align in a different manner
when free or bound (inset E), RDCs provide powerful long-range
equilibrium is described by the dissociation constant Kd. Dur-
restraints for their relative orientation in the complex when bound.
ing the titration experiment, three exchange regimes can be
observed depending on the exchange rate of the complex terface (see Fig 10B). Note however that some residues at the
formation and the chemical shift difference between the free far end of the protein may also be slightly perturbed if its
and bound states. In the slow exchange regime, two sets of global fold is affected. The intermediate regime gives rise to
signals are detected for the two states and their integral can detrimental peak broadening that may prevent the signal ob-
be used to monitor their population. From a practical point of servation: one possible work-around is a change in the ex-
view, this regime is less convenient than the fast one because perimental temperature.
the complexed signals have to be reassigned de novo. When Using CSP, Das et al. (55) have studied an antitermination
the exchange between the bound and free forms is fast as complex involved in prokaryotic transcription regulation: al-
compared with the chemical shift differences, the HSQC cor- though protein NusB is able to bind individually to a RNA
relation peaks move in a continuous manner and no new fragment, its affinity is increased by the presence of another
resonance assignment is needed. Analysis of the perturbation factor, NusE. The CSP method allowed the authors to inves-
reveals which amino acids are located at the interaction in- tigate not only binary complexes (NusB bound to NusE) but

also ternary complexes (NusB/NusE/boxA RNA). Without hav- ubiquitin by the third SH3 domain of the yeast Sla1 protein
ing to solve the complete 3D structure of this large complex, (Kd ⫽ ⬃ 40 nM) has been structurally described by NMR (60):
they were able to identify a loop in NusB that is affected by the a 15N,13C labeled SH3 domain was titrated with unlabeled
other factor NusE in the ternary complex but not in the binary ubiquitin and conversely, 15N,13C labeled ubiquitin with unla-
complex. Note that CSP can also be applied to interactions beled SH3 domain. Although more than 1500 and 1700 intra-
involving intrinsically disordered proteins (56), which are not molecular nOes have been collected for the SH3 domain and
likely to cocrystallize because of their flexibility. ubiquitin in a 1:1 complex, respectively, only 128 intermolec-
Paramagnetic probes are unique tools for studying macro- ular nOe were identified. The authors have computed NMR-
molecular complexes because of their capability to provide based structure for the complex and concluded that the SH3
long-range structural restraints (as far as 30 Å, a value to be domain binds to the canonical binding site for Pro-Rich
compared with the 5–7 Å range of nOe). Paramagnetic tags ligands.
(nitroxide radicals, Mn2⫹ chelates, or lanthanides) are intro- The CSP, PRE, and intermolecular nOe data can be used to
duced site-specifically in one of the partners (cf. Fig. 10C). map the interaction interface and model the complex starting
Two effects can be monitored on the NMR spectrum of the from the known structure of the individual partners obtained
other protein: paramagnetic relaxation enhancements (PRE) independently by X-ray diffraction or NMR. Once the various
are detected as resonance line-broadening whereas pseudo- evidences of the intermolecular interaction have been col-
contact shifts (PCS) alter the resonance frequency. The PRE lected, they can be entered into docking software that will

effects are predominantly caused by two additional relaxation predict the preferred orientation of one molecule to the other.
mechanisms (electron-nucleus dipolar or Curie-spin relax- HADDOCK developed by A. Bonvin and colleagues (61) per-
ation), which share the same dependence on the distance
forms data-driven docking using ambiguous interaction re-
from the paramagnetic center (1/r6). The paramagnetic spe-
straints (AIR). For any residue in molecule A that exhibits a
cies should be chosen with care to induce moderate broad-
CSP larger than a given threshold (called an “active” residue),
ening without washing out too many signals. Some nuclei
an ambiguous intermolecular distance between this residue
such as Gd3⫹ give large PRE but also no shifts, whereas
and any residue of molecule B is generated. The docking
others combine the two influences (57). Most lanthanides are
protocol involves a number of optimization steps including the
introduced using tags covalently bound to a thiol group in the
global positioning of the two molecules and the local refine-
protein, which should engineered by site-directed mutagene-
ment of the side-chains at the interface. Note that the
sis to contain a single Cys residue. The sketch in Fig. 10C
HADDOCK protocol can use any type of information on the
highlights the critical choice of the tag position: it should not
interaction such as site-directed mutagenesis results.
interfere with the interaction interface but should be close
Macromolecular Structure by NMR Spectroscopy—Over
enough to the partner protein.
the last decades, NMR has emerged as a technique able to
PRE has been employed to characterize a component of
provide structural information on biological molecules and
the type III secretion system in Salmonella (58): it contains
has thus been frequently compared with X-ray crystallogra-
about 120 copies of a needle protein PrgI (80 residues) and a
protein SipD (342 residues) that sits at the tip of the needle. phy. Let us briefly summarize how a structure is obtained by
The single native Cys of SipD was mutated to Ser and 14 crystallography: the beam of X-rays strikes the protein crystal
cysteine mutants were prepared for PRE measurements on producing scattered beams. The measured diffraction pattern
the smaller PrgI protein. From this extensive data set, the is converted into an electron-density map, provided that the
authors have identified two major sites where PrgI interacts phase problem has been solved. The atomic model of the
with SipD and they correlated these results with invasion protein is obtained by fitting the protein into this electron-
assays on mutants. density map. The two bottlenecks of X-ray crystallography are
Short distances are detected by means of nOe inside pro- the growth of suitable crystals and the resolution of the phase
teins and this is also applicable to protein complexes. The problem by various means such as molecular replacement or
separate expression and purification of the interaction partner the heavy atom method. The electron-density map is an im-
offer the unique opportunity of using isotope labeling to dis- age of the protein, which could be locally blurred as a result of
criminate between intra- and intermolecular contacts (one local disorder. In contrast, it is important to remember that the
protein is 13C-labeled in Fig. 10D). 13C-filtered NMR experi- NMR derived 3D representation of the protein is not an im-
ments (59) select spectroscopically intersubunit nOes. Note age of the real structure (as for X-ray) but a model of that
that for homodimeric structures, this is the only available structure that is compatible with the experimental data. In
means to characterize the dimer interface. This approach is addition, the positional uncertainty in the molecular coordi-
best suited for high-affinity complexes (Kd ⬍ ⬃ 50 nM) where nates is given by the precision and the accuracy of the model,
the two proteins remain in contact for a longer period of time. two concepts that are clearly differentiated (62). To clarify
For lower affinity, less internuclear nOes can be detected these issues, we will start with an overview of the process of
because of line broadening. For example, the recognition of protein structure determination by NMR.

With the resonance assignments in hand, one can move to cross-peaks that cannot yet be assigned unambiguously (67,
the next step, the structural characterization of the protein. It 68). Automated procedures perform well if the chemical shifts
involves collecting the largest possible number of spectral (including side-chains) have been assigned above 90% com-
parameters (primarily nOes, but also J-couplings and residual pleteness. Although their reliability in identifying peaks is
dipolar couplings): each piece of information entails an am- lower than that of a spectroscopist who visually inspects the
plitude and an assignment. Let us pinpoint this in the case of spectra, these algorithms easily compensate by the informa-
1
H-1H nOe: each visible cross-peak in a NOESY spectrum tion redundancy.
leads to an entry containing a peak amplitude and a pair of To compute a 3D structure from NMR restraints, most
assigned protons. Conceptually, nOe assignment is different software use a similar strategy, namely restrained molecular
from resonance assignment discussed earlier, though both dynamics (rMD) simulation or simulated annealing. The qual-
processes are intertwined. If two nuclei A and B have almost ifying term “restrained” indicates that the simplified force field
the same chemical shift (␯A - ␯B ⬍⬍ ␦␯A or ␦␯B, where ␦␯ is the based on the protein covalent structure (van der Waals inter-
linewidth), this results in an ambiguous nOe assignment: is C action and peptide plane planarity) is complemented by a
close to A or to B? In the absence of additional information, pseudo force field combining all NMR-derived conformational
this nOe cannot be unequivocally unraveled. The observed restraints. The conformational landscape of a protein contains
peak may also arise from the sum of two distinct interactions, numerous local minima, in which optimization algorithms
C-A and C-B. Furthermore, if all resonances have not been could be trapped. Annealing (heating and controlled cooling)

assigned, an observed nOe can potentially involve an unas- is used in metallurgy to relieve internal stresses and defects in
signed partner (D-X) and be inappropriately assigned to an- metals and optimize their mechanical properties. To over-
other nuclei (D-A). When examining an NMR based-structure, come energy barriers and converge toward a global minimum,
it is important to keep in mind the two bottlenecks: some the rMD simulation is first carried out at higher temperature
distance restraints may be either hidden because of overlaps (the atoms have high kinetic energy) and with a simplified
or misinterpreted by the spectroscopist. force field to speed up the calculation. The process is re-
In the early days of NMR, the quantification of the nOe peated from several random initial structures to sample more
cross-peaks and their conversion into distances was exten- extensively the conformational landscape. There are funda-
sively debated: could multispin effects (or spin-diffusion) mental differences between standard MD simulations and
strongly corrupt the evaluation of distance restraints (63). It is rMD simulations in the context of NMR: because of the ex-
now recognized that a qualitative assessment of distances is perimental restraints, the trajectory bears no resemblance to a
sufficient: strong cross-peaks are interpreted as a short dis- “real-life” simulation and is only used as a tool to compute a
tance (d ⬍ 3.5 Å) and weak peaks as longer distance (d ⬍ 5– 6 physically meaningful structure that satisfies the experimental
Å). In terms of precision and accuracy of resulting structure, it data. At the end of the protocol, the molecule is cooled down,
is advisable to invest more effort in a large set of qualitative a MD simulation with a complete force field (Lennard-Jones
distances than in a smaller set of precise distances. The and electrostatic interactions) in a box filled with explicit water
occurrence of a nOe cross-peak is interpreted as an upper molecules is performed.
bound restraint (d ⬍ 5– 6 Å) but its absence is seldom in- The computed structures can be envisioned as models that
cluded as a lower bound restraint (d ⬎ 7 Å): a peak might not represent our experimental data. It is then necessary to define
be visible for many reasons such as overlap or broadening the quality of the proposed structures: as the true structure is
because of conformational flexibility. As a result, only attrac- not known, evaluating the accuracy is not realizable. How well
tive experimental distance restraints that promote a compact the models agree with the NMR restraints can be assessed
folded structure are included. more easily as well as its conformity to standard features
This complicated and repetitive procedure, which involves (bond length, bond angle). Along with the bundle of structures
a substantial book-keeping effort, has been recently auto- deposited in the protein data bank (see Fig. 11), the authors
mated. The resonance assignments are used to generate will provide NMR and structure statistics (43): the number of
tentative assignments for nOe peaks in a computer program distance and dihedral constraints, the violation of these con-
that converts them into distances and generates a first bundle straints in terms of mean and standard deviation, the devia-
of structures. The software uses this first set to discard ten- tion from idealized geometry (bond length and angles), and
tative assignments that are conflicting with the majority of pairwise rmsd for the heavy atoms and the backbone. Re-
these structures and to extend the resonance assignment list. cently a suite of programs, CING (69), was presented to
A new set of structures is then computed and the cycle is validate the structural NMR ensembles at the residue level: it
repeated. This strategy is implemented in several packages: reports potential issues and directs the attention of the spec-
ARIA2 (64), CYANA (65), UNIO (ATNOS/CANDID) (66) . . . The troscopist to specific residues that deserves extensive man-
challenge for such software is to be able to cope with three ual verification.
issues: (1) incomplete or incorrect resonance assignment (pri- The NMR rmsd can be compared with the B-factors re-
marily for side-chains), (2) incorrect peak-picking, and (3) nOe ported for X-ray structures, which give an estimate of aniso-

tropic displacement of each atom about its mean position.

However, in contrast with B-factors, rmsd are only a measure
for the precision of the data. It has been reported (70) that it
overestimates the accuracy of the NMR structure ensemble
because the structure calculation procedures underestimate
the conformation freedom in proteins.
Proteins that contain disordered domain are usually difficult
(if not impossible) to crystallize and can thus be good candi-
dates for a NMR study. A number of proteins are made of
several independent globular domains connected by flexible
linkers. The linker flexibility, essential for the biological func-
tion of the protein, may prevent the growth of crystals for x-ray
diffraction. This capacity of NMR has been recently illustrated
by Gronenborn and coworkers (71) who have reported the
solution structure of a lectin in which a LysM domain is
inserted between individual repeats of a single CVNH domain.
Two flexible linkers of seven residues connect the domains

but no fixed orientation between them is derived from the
NMR data (because of lack of any nOe between the linker and
either of the domains). In such a case, NMR is able to derive
the structure of each of the domains with high precision
(rmsd ⬍ 0.25 Å on the backbone) although it is likely that false
interdomain contacts would have been detected by X-ray-
crystallography (because of crystal packing) if some crystals
could have been produced.
Protein Dynamics by NMR—The biological function of a
protein is intricately linked to its structure but also to its
dynamics. Time-dependent fluctuations in the conformation
occur during enzymatic activities, protein folding, regulation
etc. NMR is sensitive to fluctuations in many distinct time
windows ranging from picoseconds to seconds and can gen-
erally be complemented (only for fast motions) by in silico
protein dynamics simulations. The first repercussion of dy-
namics on a NMR spectrum is the resonance line-width: a
FIG. 11. NMR-based structure of B. subtilis L,D-transpeptidase small molecule exhibits narrow lines whereas large proteins
LdtBs (169 residues) (44). The antibiotic resistance found in some have broad signals. This line-broadening primarily arises from
bacterial strains for some ␤-lactam antibiotics has been assigned to the slow tumbling of large proteins but is mitigated by protein
the presence of this transpeptidase able to form unusual peptidogly- flexibility: fast internal fluctuations (⬎ ns) narrow the signals
can crosslinks. The 1H-15N HSQC spectrum of this protein is shown
in Fig. 7. Twenty structures have been computed using 3191 nOe
whereas slower motions (ms range) act in the opposite direc-
restraints, 286 dihedral restraints (J-coupling), and 169 residual dipo- tion. Besides the line-width effect, an array of NMR experi-
lar couplings. The pairwise RMSD is 0.70 Å for all heavy atoms and ments provides more detailed information on both the global
0.39 Å for the backbone. In the upper part of the figure, the best fit and internal dynamic in proteins (72).
superposition of the backbone of the 20 conformers is shown: some
Real-time NMR involves recording a series of NMR spectra
parts of the protein have been precisely characterized (␣-helices and
␤-sheets) whereas others are less well defined (loops and N- and C after initiating the process under investigation (protein folding,
termini). The disorder observed for some residues is very likely amide proton exchange, pH jump, ligand binding etc.) using a
caused by local flexibility. However, it may also be an experimental rapid-mixing apparatus. Despite the low sensitivity of NMR as
artifact because of the fortuitous lack of restraints in this area (incom- compared with fluorescence or absorbance, the time resolu-
pletely assigned residues, resonance overlaps, few neighbors within a
5 Å range, etc.). To confirm the local flexibility, NMR relaxation mea-
tion can range from 1 to 10 s. Historically limited to 1D NMR,
surements can be carried out, keeping in mind that they are only real-time NMR has gained spectral resolution with the intro-
sensitive to specific time scale motions. In the lower part of the figure, duction of fast-pulsing 2D NMR (73), where the interscan
another depiction of the transpeptidase as a ribbon representation delay is reduced in combination with selective excitation
shows the overall organization of the molecule from the same point of
pulses. With these tools, Schanda et al. (74) have studied the
view.
folding of ␣-lactalbumin (starting from a molten-globule state)

and the amide hydrogen exchange kinetics during ubiquitin parameters for the 15N-1H pair (longitudinal relaxation R1,
unfolding. transverse relaxation R2, and {1H}-15N nOe) (78) are combined
Another NMR experiment becomes more adapted for to obtain the timescale (correlation time, ␶m) and the ampli-
slower processes (10 ms to 5 s): the exchange spectroscopy tude (order parameter, S2) of the internal motion. Such a
method or EXSY (31). Although the pulse sequence is identi- separation of the internal and global motions is possible only
cal to NOESY, the spins but not only the magnetizations are if they are not correlated (79), an assumption supported by
transferred in the present case. The process (A ^ B) should their frequency difference. Note that, in contrast to chemical
be in the slow exchange regime, i.e. kex ⬍⬍ ␯A–␯B , with two exchange, NMR relaxation cannot see internal fluctuations
distinct signals visible for A and B. The 2D cross-peak inten- that are slower than the global rotation of the protein. The
sities (A3 B and B3 A) are quantified for different values of backbone dynamics can be complemented by relaxation
the exchange time T, with an upper limit set to the T1 relax- studies using side-chain probes (2H relaxation in CD3 groups
ation time of the resonances. For example, the interaction of of aliphatic side-chains) (80). When proteins are comprised of
the SH3 domain (⬃60 aa) from the Fyn tyrosine kinase with multiple domains separated by flexible linkers, their location
proline-rich peptides was studied using EXSY (75): dissocia- can be identified by 15N relaxation as shown for HscB, a 20
tion rate constants (koff) as well as thermodynamic parameters kDa cochaperone protein involved in the iron-sulfur cluster
were derived from the NMR data combined with isothermal biogenesis (81): the linker between the two domains is re-
titration calorimetry (ITC). If the exchange process is no longer vealed in the NMR study by low {1H}-15N nOes and R2/R1

in the slow exchange regime, the two lines start broadening ratios and in the crystal state by high backbone B-factors.
and then merge. Note that the spectral appearance depends Modern NMR spectroscopy offers a rich assortment of
on the kinetic parameters and not directly on the binding techniques to study protein dynamics. Some biologists have
affinity, though tighter binding yields generally longer-lived expressed criticism of NMR-derived protein dynamics be-
bound states. cause some time scales are either extremely fast or slow as
The broadening caused by microsecond to millisecond mo- compared with the biological process. One should concede
lecular motion can be exploited in the CPMG relaxation dis- that picosecond backbone fluctuations are loosely correlated
persion experiment. By applying a variable number of 180° with enzymatic reactions in the millisecond range and that
refocusing pulses, the dephasing caused by the A to B mag- real-time NMR can mainly visualize artificially slow biological
netization jumps can be suppressed: the transverse relaxation events. With these limits in mind, NMR remains the only
rate R2obs (which is the inverse of the line-width) decreases experimental technique to report protein dynamics at atomic
with more 180° pulses. For a two-site exchange model (A ^ resolution over such a wide range of time scale.
B), the dispersion profiles are governed by the rate of the NMR of Intrinsically Disordered Proteins—For many years
exchange process, the chemical shift difference and the rel- NMR has followed the footsteps of X-ray crystallography and
ative population pA and pB. This experiment remains operative focused on globular proteins composed on regular secondary
in cases with strongly skewed populations (pA ⬍⬍ pB), where structure elements. The aim of a protein NMR study was the
the minor species is nearly invisible in the NMR spectrum (76). 3D structure determination and disordered regions (linkers,
This methodology can detect low-populated excited states loops or sequence termini) were overlooked. From eukaryotic
associated with local unfolding events in proteins: the chem- genome sequencing, it has been established that more than
ical shifts of the intermediate state extracted indirectly from 30% of the proteins are comprised of disordered regions of
CPMG experiments allow its conformation to be character- more that 50 residues, while carrying out important biological
ized. CPMG relaxation has been used to investigate mutated functions (82). The amino acid composition of intrinsically
proteins, where the replacement of a single residue slightly disordered proteins (IDP) is markedly different from globular
destabilizes the global fold (75) or to protein-peptide com- counterparts with an increased content of Ala, Arg, Gly, Gln,
plexes with a small (⬃10%) mole fraction of bound peptide Ser, Glu, Lys, and Pro. Because of their intrinsic disorder,
(77). Excited invisible states are present along the folding/ IDPs cannot be crystallized and thus NMR (83) and small-
refolding pathways and in conformational changes during angle X-ray scattering (SAXS) have contributed most to their
catalysis or ligand binding. studies.
The rate at which the magnetizations relax to equilibrium The resonance assignment of IDP NMR spectra follows the
after excitation is governed by the global and internal dynam- same rules as for globular proteins but with two major differ-
ics of proteins: the overall rotational diffusion occurs in the ns ences acting in opposite directions: IDPs exhibit a compara-
time scale and the internal fluctuations on the picosecond tively restricted HN chemical shift dispersion but a more fa-
time scale. Studies of motions of N-H bonds from amide vorable line-width because of the flexibility of the polypeptide
groups provide site-specific probes for all protein residues chain. Triple resonance experiments can thus be tailored for
(except Pro). Two mechanisms contribute to the relaxation in IDPs in two ways: a better digital resolution can be achieved
the 15N-1H pair, the 15N chemical shift anisotropy and the by sampling all directions for longer time (nonuniform sam-
15
N-1H dipolar interaction discussed earlier. Three relaxation pling (84)) and the resonance can be spread in additional

dimensions in experiments with higher dimensionality (85). observed in the ␤- and ␥-synuclein, that also have been
This strategy has been successfully applied to a challenging reported to exhibit less aggregation in vitro. Such differences,
molecule, the 441-residue Tau protein: using 5D to 7D corre- which may have some implications in Parkinson’s disease,
lation experiments, this disordered protein involved in Al- can hardly be obtained by any other experimental technique.
zheimer disease has been automatically assigned (84).
CONCLUSION
Once the resonances have been assigned, it becomes pos-
sible to measure NMR parameters, keeping in mind that they NMR is currently an established tool in the field of structural
are both ensemble- and time-averaged. Chemical shifts, re- biology. Recent developments in isotopic labeling, magnet
sidual dipolar coupling, nOe, and PRE provide access to technology, electronics, and spectroscopy have pushed the
information at atomic resolution. Although thought as globally boundaries of biomolecular NMR. NMR spectral parameters
disordered, IDPs deviate locally and globally from the “ran- (J-coupling, nOe, RDC, and to a lesser extent chemical shift)
dom coil” state and exhibit local structural propensity as well provide conformational information at the atomic level on
as transient long-range contacts. An NMR investigation aims proteins. Once the NMR spectra have been assigned, these
at identifying these deviations from a random state, either angular or distance data are primary sources of information
for the free protein or on interaction with other cellular for structure computation. Molecular interactions (with a small
components. cofactor, a RNA fragment, or another proteins) can be
For globular proteins, we have seen that chemical shifts (in mapped using the same spectral parameters. In recent years,

particular 13C shifts) report on the local physicochemical en- NMR has become a major tool in the field of intrinsically
vironment: the covalent structure, the type of amino acid, and disordered proteins that are unlikely to crystallize. The dynam-
finally, on the secondary structure elements. These latter el- ics of proteins over a wide range of time scale is investigated
ements are typically transient, confined to short fragments using real-time NMR, exchange spectroscopy, or relaxation
(5–10 residues) and sparsely populated. To reliably interpret measurements. The major limitation of NMR remains the size
these small secondary shifts, it is necessary to use improved of the proteins although proteins up to 1 MDa have been
references for random coil values (nearest neighbor contribu- partially studied. Slowly, the NMR field is moving from the
tion (86)), to look at shifts from several nuclei and to combine development stage to the information-gathering stage. Solid-
them. The local structural propensity could also be charac- state NMR has not been discussed in this review but is
terized using J-couplings or short range nOes, but this ap- becoming a complementary tool especially for large soluble
proach has not been much pursued owing to the complexity multimeric proteins or membrane bound macromolecules.
of the averaging processes. In contrast, residual dipolar cou- Another emerging field of research is in-cell NMR spectros-
plings (RDC) are more promising tools as they sample an copy to delineate the role of complex and crowded cellular
angular dependence with respect to a common frame of environments on protein structure and function. In terms of
reference. Let us consider the RDC associated with a 1H-15N the number of proteins studied, NMR spectroscopy cannot
pair: because of the orientation of the NH vector, this coupling compete with other high-throughput methods in the proteom-
will change sign in a transient helical element as compared ics area but provides an alternate view on many systems of
with an unfolded chain (87). biological interest.
In the absence of preferred long-range interactions as in
Acknowledgments—We thank Dr. Adrien Favier and Dr Michael
globular proteins, long-range contacts are inherently transient Caffrey (University of Illinois at Chicago) for critically reading the
and multiple. If intramolecular contacts occur, the hydrody- manuscript, Dr. Catherine Bougault for providing NMR data, Dr.
namic radius of the protein as measured by analytical centri- Rainer Kümmerle (Bruker Fällanden-CH) for providing the NMR spec-
fugation or SAXS is expected to decrease. As far as NMR is trometer picture.
concerned, nOe are not suited to detect long-range contacts: 储 To whom correspondence should be addressed: Institut de Bi-
their range is rather limited (5–7Å) and their build-up is much ologie Structurale 41, rue Jules Horowitz, 38027 Grenoble Cedex 1 –
slower (⬎50 ms) than the life time of the contacts. In an earlier France. Tel.: (33) 4 57 42 86 98; E-mail: [email protected].
* This Tutorial is part of the International Proteomics Tutorial Pro-
section, we have seen that the dipolar interaction involving a
gramme (IPTP 16 MCP).
paramagnetic center has a much longer range (30 –35 Å):
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HUPO Tutorial

An Introduction to Biological NMR

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3006 Molecular & Cellular Proteomics 12.11

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Molecular & Cellular Proteomics 12.11 3007

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3008 Molecular & Cellular Proteomics 12.11

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disregarded otherwise. The external magnetic field B0 induces

and will thus not resonate at the same frequency. Chemical

Molecular & Cellular Proteomics 12.11 3009

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3010 Molecular & Cellular Proteomics 12.11

TABLE I than larger proteins. As a result, the detection of 4JHH and

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Molecular & Cellular Proteomics 12.11 3011

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3012 Molecular & Cellular Proteomics 12.11

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Molecular & Cellular Proteomics 12.11 3013

Despite the richness of information contained in RDC, sev-

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3014 Molecular & Cellular Proteomics 12.11

FIG. 7. 1H-15N heteronuclear single

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Molecular & Cellular Proteomics 12.11 3015

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3016 Molecular & Cellular Proteomics 12.11

or TOCSY (40) based on J-couplings (see above) and an

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Molecular & Cellular Proteomics 12.11 3017

the backbone assignment, valuable information can already

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3018 Molecular & Cellular Proteomics 12.11

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Molecular & Cellular Proteomics 12.11 3019

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3020 Molecular & Cellular Proteomics 12.11

tropic displacement of each atom about its mean position.

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Molecular & Cellular Proteomics 12.11 3021

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3022 Molecular & Cellular Proteomics 12.11

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321–325 structural an conforma-tional analysis. VCH publishers, Inc New York

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3024 Molecular & Cellular Proteomics 12.11

mics 9, 5224 –5232 J. Biomol. NMR 25, 225–234

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Molecular & Cellular Proteomics 12.11 3025

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