An Introduction to Biological NMR Spectroscopy
2013; Elsevier BV; Volume: 12; Issue: 11 Linguagem: Inglês
10.1074/mcp.o113.030239
ISSN1535-9484
Autores Tópico(s)Protein Structure and Dynamics
ResumoNMR spectroscopy is a powerful tool for biologists interested in the structure, dynamics, and interactions of biological macromolecules. This review aims at presenting in an accessible manner the requirements and limitations of this technique. As an introduction, the history of NMR will highlight how the method evolved from physics to chemistry and finally to biology over several decades. We then introduce the NMR spectral parameters used in structural biology, namely the chemical shift, the J-coupling, nuclear Overhauser effects, and residual dipolar couplings. Resonance assignment, the required step for any further NMR study, bears a resemblance to jigsaw puzzle strategy. The NMR spectral parameters are then converted into angle and distances and used as input using restrained molecular dynamics to compute a bundle of structures. When interpreting a NMR-derived structure, the biologist has to judge its quality on the basis of the statistics provided. When the 3D structure is a priori known by other means, the molecular interaction with a partner can be mapped by NMR: information on the binding interface as well as on kinetic and thermodynamic constants can be gathered. NMR is suitable to monitor, over a wide range of frequencies, protein fluctuations that play a crucial role in their biological function. In the last section of this review, intrinsically disordered proteins, which have escaped the attention of classical structural biology, are discussed in the perspective of NMR, one of the rare available techniques able to describe structural ensembles. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP 16 MCP). NMR spectroscopy is a powerful tool for biologists interested in the structure, dynamics, and interactions of biological macromolecules. This review aims at presenting in an accessible manner the requirements and limitations of this technique. As an introduction, the history of NMR will highlight how the method evolved from physics to chemistry and finally to biology over several decades. We then introduce the NMR spectral parameters used in structural biology, namely the chemical shift, the J-coupling, nuclear Overhauser effects, and residual dipolar couplings. Resonance assignment, the required step for any further NMR study, bears a resemblance to jigsaw puzzle strategy. The NMR spectral parameters are then converted into angle and distances and used as input using restrained molecular dynamics to compute a bundle of structures. When interpreting a NMR-derived structure, the biologist has to judge its quality on the basis of the statistics provided. When the 3D structure is a priori known by other means, the molecular interaction with a partner can be mapped by NMR: information on the binding interface as well as on kinetic and thermodynamic constants can be gathered. NMR is suitable to monitor, over a wide range of frequencies, protein fluctuations that play a crucial role in their biological function. In the last section of this review, intrinsically disordered proteins, which have escaped the attention of classical structural biology, are discussed in the perspective of NMR, one of the rare available techniques able to describe structural ensembles. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP 16 MCP). Nuclear magnetic resonance (NMR) 1The abbreviations used are: NMRnuclear magnetic resonance1D2D, 3D NMR, one-, two-, three-dimensional NMRCSAchemical shift anisotropyFTFourier transformHSQCheteronuclear single quantum correlation spectroscopyIDPintrinsically disordered proteinITCisothermal titration calorimetrynOenuclear Overhauser effectNOESYnuclear Overhauser effect correlation spectroscopyPREparamagnetic relaxation enhancementRDCresidual dipolar couplingr.fradio-frequency. is, at the present time, an established method in a variety of scientific fields such as physics, chemistry, biology, and medicine. However, it took more than 60 years to reach this interdisciplinary status. The discovery of nuclear magnetic resonance was made independently by two groups of prominent scientists, Felix Bloch et al. (1Bloch F. Hansen W.W. Packard M. The nuclear induction experiment.Phys. Rev. 1946; 70: 474-485Crossref Scopus (507) Google Scholar) and Edward Purcell et al. (2Purcell E.M. Torrey H.C. Pound R.V. Resonance absorption by nuclear magnetic moments in a solid.Phys. Rev. 1946; 69: 37-38Crossref Scopus (1985) Google Scholar) at the end of World War II. The 1952 Nobel Prize in Physics was awarded jointly to them "for their development of new methods for nuclear magnetic precision measurements and discoveries in connection therewith." nuclear magnetic resonance 2D, 3D NMR, one-, two-, three-dimensional NMR chemical shift anisotropy Fourier transform heteronuclear single quantum correlation spectroscopy intrinsically disordered protein isothermal titration calorimetry nuclear Overhauser effect nuclear Overhauser effect correlation spectroscopy paramagnetic relaxation enhancement residual dipolar coupling radio-frequency. Only a few years after the initial discovery, NMR entered the field of chemistry when Proctor and Yu (3Proctor W.G. Yu. F.C. The dependence of a nuclear magnetic resonance frequency upon chemical compound.Phys. Rev. 1950; 77: 717Crossref Scopus (160) Google Scholar) accidentally discovered that the two nitrogens in NH4NO3 gave rise to two different signals. This first observation of the chemical shift was confirmed one year later by the detection of three lines in the spectrum of ethanol. In 1952 the first commercial Varian NMR spectrometer operating at 30 MHz for 1H was produced. In 1953, Overhauser (4Overhauser A.W. Polarization of nuclei in metals.Phys. Rev. 1953; 92: 411-415Crossref Scopus (1153) Google Scholar) observed that the saturation of electrons in metals led to an increase of the nuclear polarization: this effect known later as the "nuclear Overhauser effect" was the first evidence that spins (nuclei or electrons) could communicate through some spin–spin interactions. These double resonance methods were also used to detect spin–spin coupling, the other types of interaction between nuclei and in 1961, Freeman and Whiffen (5Freeman R. Whiffen D.H. Determination of the relative signs of proton spin coupling constants by double irradiation.Mol. Phys. 1961; 4: 321-325Crossref Scopus (29) Google Scholar) analyzed the spin–spin coupling network in 2-furoic acid. In these early years, NMR was a rather insensitive method: for instance, pure liquids were required to detect 13C NMR spectra. Stronger electromagnets were designed to reach 100 MHz for the 1H frequency until the emergence of superconducting magnets in the early 1960s. The first 200 MHz spectrum of ethanol (6Nelson F.A. Weaver H.E. Nuclear magnetic resonance spectroscopy in super-con-ducting magnetic fields.Science. 1964; 146: 223-232Crossref PubMed Scopus (17) Google Scholar) was published in 1964 after solving a great deal of technical challenges such as magnet homogeneity and stability. A further gain in sensitivity was provided by the introduction of Fourier transformed (FT) NMR (7Ernst R.R. Anderson W.A. Application of Fourier transformed spectroscopy to magnetic resonance.Rev. Sci. Instrum. 1966; 37: 93-102Crossref Scopus (1271) Google Scholar) in 1966 by Ernst and Anderson, both working at Varian. The ability to excite simultaneously and then unravel all signals was a methodological breakthrough that opens the door to the development of numerous pulse sequences. In 1957, exploratory studies were undertaken on small biological molecules such as common amino-acids and the first spectrum of bovine pancreatic ribonuclease (8Saunders M. Wishnia A. Kirkwood J.G. The nuclear magnetic resonance spectrum of ribonuclease.J. Am. Chem. Soc. 1957; 79: 3289-3290Crossref Scopus (92) Google Scholar) was recorded at 40 MHz. After failing to observe a spectrum in H2O, these authors reported a 1H spectrum in D2O that exhibited four lines corresponding to the various types of protons (aromatic and aliphatic). Most of the research in the 1960s was carried out on synthetic or natural peptides and on some paramagnetic proteins such as cytochrome c and myoglobin, where some resonances fall outside of the standard range of chemical shift. The greatest hurdle was the suppression of the water signal that is several orders of magnitude larger than the signal of interest. The next step was the introduction of two-dimensional (2D) NMR by R. Ernst et al. in 1976 following a clever idea of J. Jeener, a Belgian physicist. The introduction of an additional frequency axis led to correlation maps (9Aue W.P. Bartholdi E. Ernst R.R. Two-dimensional spectroscopy. Application to nuclear magnetic resonance.J. Chem. Phys. 1976; 64: 2229-2246Crossref Scopus (3089) Google Scholar) between spins (either via J-coupling or nOe) and to powerful tools for resonance assignment. Today, NMR is very unique in the versatility of the multidimensional experiments that can be implemented. In 1991, the Nobel Prize in Chemistry was awarded to Richard Ernst "for his contributions to the development of the methodology of high resolution nuclear magnetic resonance (NMR) spectroscopy." 2D NMR was quickly transferred to the field of biomolecules by the group of K. Wüthrich. Its feasibility was demonstrated on a 10 mm sample of BPTI, a 58 amino acid protein, despite the then-available limited computational facilities. The very high protein concentration, that drastically limited the use of this new method at that time, has been greatly reduced over the years as a result of numerous technical improvements. In 1985, the first structure of a small globular protein was published (10Williamson M.P. Havel T.F. Wüthrich K. Solution conformation of proteinase inhibitor IIA from bull seminal plasma by 1H nuclear magnetic resonance and distance geometry.J. Mol. Biol. 1985; 182: 295-315Crossref PubMed Scopus (530) Google Scholar) but for the well-established community of X-ray crystallographers, the reaction was disbelief, claiming that the obtained structure had been modeled using other previously crystallized proteins. The credibility of NMR as structural tool for proteins was strengthened over the years as its performance increased: 3D NMR was introduced first on unlabeled proteins followed quickly by a new set of triple resonance experiments (11Bax A. Multidimensional nuclear magnetic resonance methods for protein studies.Curr Opin Struc Biol. 1994; 4: 738-744Crossref Scopus (192) Google Scholar) using 15N and 13C labeled samples. In 2002, The Nobel Prize in Chemistry was awarded to Kurt Wüthrich "for his development of nuclear magnetic resonance spectroscopy for determining the three-dimensional structure of biological macromolecules in solution." NMR spectrometers devoted to structural biology benefit from several recent technological achievements: (1) higher magnetic field (≥ 950 MHz) can be reached using new superconducting material, (2) cryoprobes, in which the transmit/receive coils are maintained at low temperature to reduce the noise, have become standard equipment, (3) the design of the spectrometer electronics leads to superb experimental long-term stability, and (4) alternate processing methods are possible with the increased power of computers. Fig. 1 shows a recent NMR spectrometer at intermediate field (600 MHz): most biological studies can be carried out on this midrange model and could be completed by getting access to a large-scale facility (> 950 MHz). In recent years, biological NMR has evolved toward more diverse applications. As depicted in Fig. 2, the number of published structures solved by NMR has stagnated over the years in comparison with the structures solved by X-ray diffraction. This trend can easily be explained by the fact that solving a protein structure by X-ray can be quite fast once suitable crystals have been obtained. However, NMR can provide other types of information that is hardly amenable by crystallography: dynamics can be investigated by NMR over a wide range of time scales (12Mittermaier A.K. Kay L.E. Observing biological dynamics at atomic resolution using NMR.Trends Biochem. Sci. 2009; 34: 601-611Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar), from slow exchange where the two interconverting species are visible to fast motion using relaxation measurements. In the field of drug discovery (13Pellecchia M. Bertini I. Cowburn D. Dalvit C. Giralt E. Jahnke W. James T.L. Homans S.W. Kessler H. Luchinat C. Perspectives on NMR in drug discovery: a technique comes of age.Nature Reviews Drug Discovery. 2008; 7: 738-745Crossref PubMed Scopus (325) Google Scholar), chemical shift mapping provides information on which part of the protein is interacting with the ligand and NMR is very powerful at screening or optimizing hits. In conclusion, the ecological niche of NMR is currently not restricted to protein structure determination but covers a wider range of relevant information. A NMR spectrum can only be observed for nuclei that possess a net spin. In this respect, the most abundant nucleus in a protein, hydrogen, is well suited as its most abundant isotope (1H) has spin ½. In contrast, carbon, nitrogen, and oxygen are not easily visible by NMR, at least for their most abundant isotopes (12C, 14N, and 16O). We will discuss later in this review how to enrich the protein with isotopes ("isotope-labeling") such as 13C and 15N. Although these strategies were very expensive two decades ago, uniform or selective labeling is now cost-effective. NMR experiments are carried out in a static magnetic field B0 (several Tesla) aligned conventionally along the +z axis. As a result of this field, the space is no longer isotropic and all interactions experienced by the spins will depend on the orientation of the molecule with respect to the magnetic field B0. In mathematical terms, the anisotropic NMR interactions are described by second-rank tensors or 3 × 3 matrices. However, in liquid state NMR, the molecule under investigation is rotating freely with a correlation time τc (1–50 ns) much smaller than the acquisition time: if this rotation is isotropic, all interactions will average out and only the isotropic component will be observed. This explains the sharpness of resonance typically seen in solution NMR spectra as compared with solid-state spectra. The atomic-resolution power of NMR is intrinsically linked to the occurrence of chemical shift. In a NMR spectrum, the magnitude or intensity of the resonance is displayed along a single frequency axis (in the case of 1D NMR) or several axes (for multidimensional NMR). Chemical shift is usually expressed not in Hz but in ppm relative to a standard: δ(ppm)=106⋅v−v0v01 where ν is the signal frequency in Hz and ν0 that of a reference compound. Thus, chemical shifts in ppm can be compared between data sets recorded at different field strength. Several calibration standards are available: tetramethylsilane (TMS) is used in organic solvents but because of its poor solubility in water, it is replaced by 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS) for protein NMR (IUPAC recommendation). However, to avoid any additional compound that might interfere with the protein, most spectroscopists use, as a calibration intermediate, the water line although its position is temperature- and pH-dependent. Measuring chemical shift value is the most amenable task of NMR spectroscopy. The wealth of information provided by chemical shift data depends on the availability of the individual resonance assignments. If the chemical shifts of compound A change when compound B is added to the sample, we already know that A and B are interacting. If the resonances of A have been assigned (see below), then these changes can be interpreted at the atomic level. Through such an experiment applied to a protein-ligand interaction (13Pellecchia M. Bertini I. Cowburn D. Dalvit C. Giralt E. Jahnke W. James T.L. Homans S.W. Kessler H. Luchinat C. Perspectives on NMR in drug discovery: a technique comes of age.Nature Reviews Drug Discovery. 2008; 7: 738-745Crossref PubMed Scopus (325) Google Scholar), we can learn what parts of the small molecule are interacting and to which part of the macromolecular target the small molecule is bound. Chemical shift is by essence an anisotropic interaction but we only observe the isotropic part in solution. At high field, chemical shift anisotropy (CSA) can broaden NMR signals for some nuclei (CO in proteins for example) but it can be safely 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: B→loc=B→0+B→ind 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-identified partners: its prediction or quantitative interpretation is thus more complex. Let us consider the chemical shifts of backbone 15N in proteins: the standard chemical shift range for this nucleus runs from about 100 to 135 ppm, but outliers at 77.1 and 142.81 ppm have been reported. In one of the largest (723 residues) assigned proteins, Malate Synthase G, 71 alanines have been assigned: 4 Ala 15N exhibit a shift above 130 ppm and 4 below 118 ppm. This clearly shows that a signal cannot be assigned on the basis of the covalent structure of the protein. As the number of assigned proteins is increasing, greater insights have been gained into the contribution to chemical shift of torsion angles, aromatic rings (Fig. 3), solvent accessibility, temperature, pH, and ionic strength. Several databases are available over the internet as chemical shift repositories: the largest one is the BioMag-ResBank (http://www.bmrb.wisc.edu), which contains 7800 entries (as of 2012). Smaller curated databases, where the data found in the BioMag-ResBank have been selected and corrected, have also been generated such as TALOS or TALOS+ (14Shen Y. Delaglio F. Cornilescu G. Bax A. TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts.J. Biomol. NMR. 2009; 44: 213-223Crossref PubMed Scopus (2012) Google Scholar) for more specific purposes. For each type of amino acid, chemical shifts can be interpreted in terms of secondary structure by subtracting reference values for random coil structures. Data obtained in the 1970s on 1H shifts on small peptides Gly-Gly-Xaa-Ala (15Bundi A. Wüthrich K. 1H-NMR parameters of the common amino acid residues measured in aqueous solutions of the linear tetrapeptides H-Gly-Gly-X-L-Ala-OH,".Biopolymers. 1979; 18: 285-297Crossref Scopus (830) Google Scholar) have been recently supplemented by 13C and 15N data in various aqueous and organic solvent conditions (16Wishart D.S. Interpreting protein chemical shift data.Prog Nucl Mag Res Sp. 2011; 58: 62-87Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar) and are available at the BioMag-ResBank. These random coil values can be further improved by integrating nearest-neighbor effects. Beside random coil values, reference values for α-helices and β-sheet (17Zhang H. Neal S. Wishart D.S. RefDB: a database of uniformly referenced protein chemical shifts.J. Biomol. NMR. 2003; 25: 173-195Crossref PubMed Scopus (373) Google Scholar) have been assembled from NMR data for each residue type in experimentally observed secondary structure. For the 15N shift in Ala, a reference value of 121.4 ppm is found in α-helices, 124.5 in β-sheet and 123.6 in random coils. As far as carbons are concerned, the Cα and CO move to higher chemical shifts in α-helices and to lower shifts in β-strands but the trend is reversed for the Cβ resonances. This observation is the basis of the Chemical Shift Index (CSI) (18Wishart D.S. Sykes B.D. Richards F.M. The chemical shift index: a fast and simple method for the assignment of protein secondary structure through NMR spectroscopy.Biochemistry. 1992; 31: 1647-1651Crossref PubMed Scopus (2014) Google Scholar, 19Wishart D.S. Sykes B.D. The 13C chemical-shift index: a simple method for the identification of protein secondary structure using 13C chemical-shift data.J. Biomol. NMR. 1994; 4: 171-180Crossref PubMed Scopus (1908) Google Scholar), a method that uses chemical shifts to identify the type and location of protein secondary structures along a protein chain. As compared with circular dichroism (CD) spectra that are used to determine the global protein secondary structure content, the CSI method provides information at the residue level. Without resource to nOe measurements (see below) and structure computation, the secondary structure of proteins can be obtained from chemical shifts. Along the same lines, the chemical shifts can also be used to directly derive torsion angles. The backbone conformation is defined by two dihedral angles (φ and ψ) for each amino acid as well as several angles for the side-chain (χ1, χ2…). TALOS uses a database of protein sequences, chemical shifts and dihedral angles to predict backbone dihedral angles, but fails to make any prediction only for roughly 30% of the residues. The success of the TALOS (20Cornilescu G. Delaglio F. Bax A. Protein backbone angle restraints from searching a database for chemical shift and sequence homology.J. Biomol. NMR. 1999; 13: 289-302Crossref PubMed Scopus (2738) Google Scholar) methods (and its improved version TALOS+) is clearly illustrated by the high number of citations of the original paper (> 2000 citations). Ongoing research is currently aimed at computing protein structures using only chemical shift information: the goal of this strategy, which can immediately follow the resonance assignment, is to evade the lengthy process of nOe assignment (see below). This approach makes use of the Rosetta algorithm for de novo protein modeling. This algorithm builds a large number of models for the protein on the basis of fragments from the PDB database that share some sequence similarity: only the models that are compact and energetically favorable are retained. In the CS-Rosetta approach (21Shen Y. Lange O. Delaglio F. Rossi P. Aramini J.M. Liu G. Eletsky A. Wu Y. Singarapu K.K. Lemak A. Ignatchenko A. Arrowsmith C.H. Szyperski T. Montelione G.T. Baker D. Bax A. Consistent blind protein structure generation from NMR chemical shift data.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 4685-4690Crossref PubMed Scopus (688) Google Scholar), backbone chemical shifts are used to select suitable fragments: with this additional information, the convergence of this Monte-Carlo algorithm requires a smaller number of models and thus smaller amounts of computing time, at least for proteins of relatively simple topology. Scalar coupling (or J-coupling) is a through-bond interaction between two nuclei (A and X) with a nonzero spin. It is an indirect interaction between the two spins that is mediated by the electrons: one spin perturbs the spins of the shared electrons, which in turn will perturb the second spin. Only the isotropic part of the anisotropic interaction is detected in liquid-state NMR. Reported in Hz, it is field-independent and causes NMR signals to be split in multiple peaks: if two spins ½ are scalar coupled, the spectrum of each will be a doublet (see Fig. 4) and the separation between the two lines is the coupling constant JAX. The presence of two lines can be understood as two distinct populations of spin A: the spins A, which have a neighbor X in the "up" spin state (↑) (i.e. aligned along the magnetic field +z), will resonate at δ + ½JAX whereas the spins A, which have a neighbor X in the "down" spin state (↓), resonate at δ - ½JAX. The indirect interaction may either increase or decrease the resonance frequency: the absolute sign of a J-coupling cannot be experimentally determined by NMR, but only the relative sign of two couplings sharing a common nucleus. Scalar couplings are denoted as nJAX, in which A and X are the interacting nuclei and n the number of covalent interceding bonds. One-bond coupling (1J) are an order of magnitude larger than two- and three-bond couplings (2J, 3J), which in turn are larger than long-range coupling such as 4J and 5J (22Bystrov V.F. Spin-spin coupling and the conformational states of peptides systems.Prog. Nucl. Mag. Res. Sp. 1976; 10: 41-81Abstract Full Text PDF Scopus (953) Google Scholar). Typical values for couplings observable in proteins are reported in Table I.Table ITypical values for the scalar coupling in proteins2JHH9–15 Hz1JNCO15 Hz3JHH0–14 Hz1JCαCO55 Hz1JNH90 Hz1JCαCβ35 Hz1JNCα7–11 Hz1JCH (aliphatic)130–150 Hz2JNCα4–9 Hz1JCH (aromatic)160 Hz Open table in a new tab For experimental purposes, the magnitude of any scalar coupling should always be compared with the line-width (Δν) of the associated signals. A coupling smaller than the line-width is hardly visible on the 1D NMR spectrum and a 2D correlation experiment through this coupling will have a low efficiency and thus poor sensitivity. As discussed below, the NMR line width increases with the size and rigidity of the molecule and small peptides exhibit much narrower signals than larger proteins. As a result, the detection of 4JHH and 5JHH couplings, which is straightforward in peptides, becomes unrealistic on a 20 kDa protein. By comparing the magnitude of the 1H-1H coupling 2JHH and 3JHH with that of the heteronuclear one (1JNH, 1JCH …) (see Table I), one 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 larger heteronuclear couplings. As scalar couplings stem from the bond orbitals, they all contain structural information. One-bond couplings show little variation for a type of spin pairs: however, 1JCH for aliphatic carbons (sp3) are smaller than for aromatic carbons (sp2) and for each hybridization, a rough correlation with the 13C chemical shift has been reported (23Zwahlen C. Legault P. Vincent S.J.F. Greenblatt J. Konrat R. Kay L.E. Methods for measurement of intermolecular NOEs by multinuclear NMR spectroscopy: application to a bacteriophage ë N-peptide/boxB RNA complex.J. Am. Chem. Soc. 1997; 119: 6711-6721Crossref Scopus (537) Google Scholar). Bax and coworkers (24Vuister G.W. Delaglio F. Bax A. An empirical correlation between 1JCaHa and protein backbone conformation.J. Am. Chem. Soc. 1992; 114: 9674-9675Crossref Scopus (73) Google Scholar) have analyzed the variation of the 1JCαHα in proteins [135 Hz -150 Hz] and reported a empirical correlation with the backbone dihedral angles (φ and ψ) of the residue. With this limited variation of the 1J, heteronuclear correlation experiments (such as HSQC or HMQC) could be designed to yield 1H-X cross-peaks of homogeneous amplitude. Similarly, 2J couplings (2JCαN, 2JHNCα …) show empirical correlations with φ and ψ angle but the difficulty lies in the interpretation of the simultaneous dependences on more than a single torsion angle (25Schmidt J.M. Hua Y. Löhr F. Correlation of 2J couplings with protein secondary structure.Proteins. 2010; 78: 1544-1562PubMed Google Scholar). By far, the most valuable structural information is derived from three-bond mediated vicinal couplings (3J): in the early 1960s Martin Karplus (26Karplus M. Vicinal proton coupling in nuclear magnetic resonance.J. Am. Chem. Soc. 1963; 85: 2870-2871Crossref Scopus (2288) Google Scholar) established a relationship between the dihedral (torsion) angle (Φ) between protons (H-C–C-H) and vicinal coupling 3J. The general form of the Karplus relationship is: 3J(Φ)=A cos2(Φ)+B cos (Φ)+C2 and the coefficients A, B, and C are parameterized for each combination of nuclei. In proteins, the 3JHNHα coupling provides information on the φ backbone angle whereas the 3JHαHβ provides information on the side-chain χ1 angle (21Shen Y. Lange O. Delaglio F. Rossi P. Aramini J.M. Liu G. Eletsky A. Wu Y. Singarapu K.K. Lemak A. Ignatchenko A. Arrowsmith C.H. Szyperski T. Montelione G.T. Baker D. Bax A. Consistent blind protein structure generation from NMR chemical shift data.Proc. Natl. Acad. Sci. U.S.A. 2008; 105: 4685-4690Crossref PubMed Scopus (688) Google Scholar). In α-helices (φ = −64° ± 7°), a small 3JHNHα is observed (< 4 Hz) whereas for β-sheets (φ = −120° ± 10°) larger couplings are present (> 4 Hz). Unfortunately, no 3JHH coupling is linked to the other backbone angle ψ, which can be obtained in a 15N labeled peptide using the much smaller 3JHαN. The β-methylene moiety found in most amino-acids is a prochiral center, i.e. it could become a chiral center by replacing one of the two protons by another group (a deuterium for instance). As a result, the pro-R and the pro-S protons have different chemical shifts. Their stereospecific assignment is achieved by combining several vicinal coupling constants (3JHαHβ1, 3JHαHβ2, 3JNHβ1, 3JC'Hβ1 …) and several distance measurements based on nOe information (see below). Similarly, the two CH3 in Leu and Val isopropyl groups need to be stereospecifically assigned. It has been shown that the availability of stereospecific assignment for these prochiral centers improves the accuracy and the precision of the derived NMR structures (27Güntert P. Braun W. Billeter M. Wüthrich K. Automated stereospecific proton NMR assignments and their impact on the precision of protein structure determinations in solution.J. Am. Chem. Soc. 1989; 111: 3997-4004Crossref Scopus (240) Google Scholar). How could a scalar coupling be evidenced
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