Determination of protein global folds using backbone residual dipolar coupling and long-range NOE restraints

We report the determination of the global fold of human ubiquitin using protein backbone NMR residual dipolar coupling and long-range nuclear Overhauser effect (NOE) data as conformational restraints. Specifically, by use of a maximum of three backbone residual dipolar couplings per residue (N_i-H^N _i, N_i-C_i–1, H^N _i - C_i–1) in two tensor frames and only backbone H^N-H^N NOEs, a global fold of ubiquitin can be derived with a backbone root-mean-square deviation of 1.4 Å with respect to the crystal structure. This degree of accuracy is more than adequate for use in databases of structural motifs, and suggests a general approach for the determination of protein global folds using conformational restraints derived only from backbone atoms.     

Solution structure of T4moC,the Rieske ferredoxin component of the toluene 4-monooxygenase complex

Toluene 4-monooxygenase, a four-protein complex from Pseudomonas mendocina KR1, catalyzes the NADH- and O₂-dependent hydroxylation of toluene to form p-cresol. The solution structure of the 112-amino-acid Rieske ferredoxin component, T4moC, was determined from 2D and 3D ¹H, ¹³C, and ¹⁵N NMR data. The structural model was refined through simulated annealing by molecular dynamics in torsion angle space with input from 1650 experimental restraints, including 1264 inter-proton distance restraints obtained from NOEs, 247 non-redundant intra-residue NOEs, 26 hydrogen bond restraints, and 113 dihedral angle (, ) restraints. The 20 calculated conformers that best satisfied the input restraints were submitted to refinement in explicit solvent to improve the stereochemical quality. With exclusion of ill-defined N- and C-terminal segments (Ser2; His111–Ser112) and residues near to the [2Fe-2S] cluster, the atomic root mean square deviation for the 20 conformers with respect to the mean coordinates was 1.09 Å for the backbone and 1.60 Å for all non-hydrogen atoms. The T4moC structure consists of 10 -strands arranged in the three anti-parallel -sheet topology observed in all Rieske [2Fe-2S] domain proteins. The S of Cys45 and Cys64 and the N¹ of His47 and His67 provide the ligands to the [2Fe-2S] cluster of T4moC. ¹H–¹⁵N HSQC measurements show that both His47-N² and His67-N² are protonated at the pH of the NMR experiments. Comparisons are made between the present NMR structure, previous paramagnetic NMR studies of T4moC, and the X-ray structures of other members of the Rieske protein family.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00775-004-0594-4     

Solution structure determination of the two DNA-binding domains in the Schizosaccharomyces pombe Abp1 protein by a combination of dipolar coupling and diffusion anisotropy restraints

We have solved the solution structure of the N-terminal region of the fission yeast centromere protein, Abp1, bound to a 21-base pair DNA fragment bearing its recognition site (Mw = 30 kDa). Although the two DNA-binding domains in the Abp1 protein were defined well by a conventional NOE-based NMR methodology, the overall structure of the Abp1 protein was poorly defined, due to the lack of interdomain distance restraints. Therefore, we additionally used residual dipolar couplings measured in a weakly aligned state, and rotational diffusion anisotropies. Neither the NH residual dipolar couplings nor the backbone ¹⁵N T ₁/T ₂ data were sufficient to determine the overall structure of the Abp1 protein, due to spectral overlap. We used a combination of these two orientational restraints (residual dipolar coupling and rotational diffusion anisotropy), which significantly improved the convergence of the overall structures. The range of the observed T ₁/T ₂ ratios was wider (20–50 for the secondary structure regions of Abp1) than the previously reported data for several globular proteins, indicating that the overall shape of the Abp1DNA complex is ellipsoid. This extended form would facilitate the recognition of the two separate sites in the relatively long DNA sequence by the DNA-binding domains of Apb1.     

The solution structure of a gallium-substituted putidaredoxin mutant: GaPdx C85S

The Fe₂S₂ cluster of the ferredoxin putidaredoxin (Pdx) can be replaced by a single gallium ion, giving rise to a colorless, diamagnetic protein in which, apart from the metal binding site, the major structural features of the native ferredoxin are conserved. The solution structure of the C85S variant of gallium putidaredoxin (C85S GaPdx), in which a non-ligand cysteine is replaced by a serine, has been determined via multidimensional NMR methods using uniformly ¹⁵N,¹³C labeled samples of C85S GaPdx. Stereospecific assignments of leucine and valine methyl resonances were made using ¹³C,¹H HSQC spectra obtained with fractionally ¹³C-labeled samples, and backbone dihedral angle restraints were obtained using a combination of two-dimensional J-modulated ¹⁵N,¹H HSQC and three-dimensional (HN)CO(CO)NH experiments. A total of 1117 NOE-derived distance restraints were used in the calculations, including 454 short range ($i - j 3$), 456 long range (i - j 4) interresidue restraints and 207 non-trivial intraresidue restraints. 97 and 55 ₁ angular restraints were also included in the calculation of a family of 20 structures using a combined distance geometry-simulated annealing protocol. Most regions of the protein are well defined in the calculations, with an RMSD of 0.525 Å for backbone atoms excluding the metal binding loop (residues 34–48) and the last three C-terminal residues (residues 103–106). Where comparison is possible, these regions show an increase in dynamic behavior over the native protein, as does the loop containing residues 74–76. Structural and dynamic differences between native Pdx and GaPdx are discussed in relation to charge and packing of the metal binding site.     

The three-dimensional structure of acyl-coenzyme A binding protein from bovine liver: Structural refinement using heteronuclear multidimensional NMR spectroscopy

Summary The 3D structure of bovine recombinant acyl-coenzyme A binding protein has been determined using multidimensional heteronuclear magnetic resonance spectroscopy in a study that combines investigations of ¹⁵N-labeled and unlabeled protein. The present structure determination is a refinement of the structure previously determined (Andersen, K.V. and Poulsen, F.M. (1992) J. Mol. Biol., 226, 1131–1141). It is based on 1096 distance restraints and 124 dihedral angle restraints of which 69 are for -angles and 8 for chiral centers and 47 for prochiral centers. The new experimental input for the structure determination has provided an increase of 263 distance restraints, 5 -angle restraints, and 32 -angle restraints in 2 chiral centers, and 31 prochiral centers restraining an additional 23 ¹, 8 ², and 1 ³ angles. The increase of 300 distance and dihedral angle restraints representing an additional 30% of input parameters for the structure determination has been shown to be in agreement with the first structure. A set of 29 structures has been calculated and each of the structures has been compared to a mean structure to give an atomic root mean square deviation of 0.44±0.12 (1 is 0.1 nm) for the backbone atoms C, C, and N in the four -helices A1, residues 4–15, A2, residues 21–36, A3, residues 51–62 and A4, residues 65–84. The loop-region of residues Gly⁴⁵-Lys⁵⁰ could not be defined by the restraints obtained by NMR.The program PRONTO has been used for the spectrum analysis, assignment of the individual nuclear Overhauser effects, the integration of the cross peaks, and the measurement of the coupling constants. The programs DIANA, X-PLOR and INSIGHT have been used in the structure calculations and evaluations.     

Probing hydrogen bonds in the antibody-bound HIV-1 gp120 V3 loop by solid state NMR REDOR measurements

We describe solid state NMR measurements on frozen solutions of the complex of the 24-residue HIV-1 gp120 V3 loop peptide RP135 with the Fab fragment of the anti-gp120 antibody 0.5, using rotational echo double resonance (REDOR). In order to probe possible hydrogen bonding between arginine side chains and glycine backbone carbonyls in the region of the conserved Gly-Pro-Gly-Arg (GPGR) motif of the V3 loop, RP135 samples were prepared with ¹⁵N labels at the nitrogen positions of arginine side chains and ¹³C labels at glycine carbonyl positions and ¹³C-detected ¹³C-¹⁵N REDOR measurements were performed on peptide/antibody complexes of these labeled samples. Such hydrogen bonding was previously observed in a crystal structure of the V3 loop peptide/antibody complex RP142/59.1 [Ghiara et al. (1994) Science, 264, 82–85], but is shown by the REDOR measurements to be absent in the RP135/0.5 complex. These results confirm the antibody-dependent conformational differences in the GPGR motif suggested by previously reported solid state NMR measurements of and backbone dihedral angles in the RP135/0.5 complex. In addition, we describe REDOR measurements on the helical synthetic peptide MB(i+4)EK in frozen solution that establish our ability to detect ¹³C-¹⁵N dipole–dipole couplings in the distance range appropriate to these hydrogen bonding studies. We also report the results of molecular modeling calculations on the central portion RP135, using a combination of the solid state NMR restraints of Weliky et al. [Nat. Struct. Biol., 6, 141–145, 1999] and the liquid state NMR restraints of Tugarinov et al. (Nat. Struct. Biol., 6, 331–335, 1999]. The dynamics calculations demonstrate the mutual compatibility of the two sets of experimental structural restraints and reduce ambiguities in the solid state NMR restraints that result from symmetry and signal-to-noise considerations.     

The relative orientation of the fibronectin 6F11F2 module pair: A 15N NMR relaxation study

The structure of a pair of modules (⁶F1¹F2), that forms part of the collagen-binding region of fibronectin, is refined using heteronuclear relaxation data. A structure of the pair was previously derived from ¹H-¹H NOE and ³ J _HHN data [Bocquier et al. (1999) Structure, 7, 1451–1460] and a weak module–module interface, comprising Leu19 and Leu28, in ⁶F1, and Tyr68 in ²F1, was identified. In this study, the definition of the average relative orientation of the two modules is improved using the dependence of ¹⁵N relaxation on rotational diffusion anisotropy. This structure refinement is based on the selection of a subset of structures from sets calculated with NOE and ³ J _HHN data alone, using the quality of the fits to the relaxation data as the selection criterion. This simple approach is compared to a refinement strategy where ¹⁵N relaxation data are included in the force field as additional restraints [Tjandra et al. (1997) Nat. Struct. Biol., 4, 443–449].     

Three-dimensional structure of the Hck SH2 domain in solution

The hematopoietic cellular kinase (Hck) is a member of the Srcfamily of non-receptor protein-tyrosine kinases that is expressedpredominantly in granulocytes, monocytes and macrophages. Recentobservations suggest that Hck may be activated in HIV-infected macrophagesand in chronic myelogenous leukemia cells that express Bcr-Abl. In order toincrease our understanding of the structural basis for regulation of Hckactivity under normal and pathological conditions, we have solved thesolution structure of the uncomplexed Hck SH2 domain using NMR spectroscopy.A novel procedure that uses intraresidueH^NH distances as references forconverting NOE intensities into distance restraints has been described. Atotal of 1757 significant experimental restraints were derived from NMRspectroscopic data including 238 medium-range and 487 long-range distancerestraints and 177 torsion angle restraints. These restraints were used in asimulated annealing procedure to generate 20 structures with the programDYANA. Superimposition of residues 5–104 upon the mean coordinate setyielded an average atomic rmsd values of 0.42 ± 0.08 Å for theN,C,C atoms and 0.81 ± 0.08 Å forall heavy atoms. Rmsd values for those residues in the regions of orderedsecondary structure were 0.27 ± 0.04 Å for theN,C,C atoms and 0.73 ± 0.06 Å forall heavy atoms.     

An approach to global fold determination using limited NMR data from larger proteins selectively protonated at specific residue types

Summary A combination of calculation and experiment is used to demonstrate that the global fold of larger proteins can be rapidly determined using limited NMR data. The approach involves a combination of heteronuclear triple resonance NMR experiments with protonation of selected residue types in an otherwise completely deuterated protein. This method of labelling produces proteins with -specific deuteration in the protonated residues, and the results suggest that this will improve the sensitivity of experiments involving correlation of side-chain (¹H and ¹³C) and backbone (¹H and ¹⁵N) amide resonances. It will allow the rapid assignment of backbone resonances with high sensitivity and the determination of a reasonable structural model of a protein based on limited NOE restraints, an application that is of increasing importance as data from the large number of genome sequencing projects accumulates. The method that we propose should also be of utility in extending the use of NMR spectroscopy to determine the structures of larger proteins.The first two authors contributed equally to this work.     

Backbone dynamics of the human CC-chemokine eotaxin

Eotaxin is a CC chemokine with potent chemoattractant activity towards eosinophils. ¹⁵N NMR relaxation data have been used to characterize the backbone dynamics of recombinant human eotaxin. ¹⁵N longitudinal (R₁) and transverse (R₂) auto relaxation rates, heteronuclear ¹H-¹⁵N steady-state NOEs, and transverse cross-relaxation rates (_xy) were obtained at 30 °C for all resolved backbone secondary amide groups using ¹ H-detected two-dimensional NMR experiments. Ratios of transverse auto and cross relaxation rates were used to identify NH groups influenced by slow conformational rearrangement. Relaxation data were fit to the extended model free dynamics formalism, yielding parameters describing axially symmetric molecular rotational diffusion and the internal dynamics of each NH group. The molecular rotational correlation time (_m) is 5.09±0.02 ns, indicating that eotaxin exists predominantly as a monomer under the conditions of the NMR study. The ratio of diffusion rates about unique and perpendicular axes (D/D) is 0.81±0.02. Residues with large amplitudes of subnanosecond motion are clustered in the N-terminal region (residues 1–19), the C-terminus (residues 68–73) and the loop connecting the first two -strands (residues 30–37). N-terminal flexibility appears to be conserved throughout the chemokine family and may have implications for the mechanism of chemokine receptor activation. Residues exhibiting significant dynamics on the microsecond–millisecond time scale are located close to the two conserved disulfide bonds, suggesting that these motions may be coupled to disulfide bond isomerization.     

Determination of the glycosidic torsion angles in uniformly 13C-labeled nucleic acids from vicinal coupling constants 3J(C2)/4-H1' and 3J(C6)/8-H1'

A two-dimensional, quantitative J-correlation NMR experiment for precise measurements of the proton-carbon vicinal coupling constants ³J_C2/4–H1 and ³J_C6/8–H1 in uniformly ¹³C-labeled nucleic acids is presented. To reduce loss of signal due to ¹H -¹³C dipole-dipole relaxation, a multiple-quantum constant time experiment with appropriately incorporated band selective ¹H and ¹³C pulses was applied. The experiment is used to obtain the ³J_C2/4–H1 and ³J_C6/8–H1 coupling constants in a uniformly ¹³C, ¹⁵N-labeled [d(G₄T₄G₄)]₂ quadruplex. The measured values and glycosidic torsion angles in the G-quadruplex, obtained by restrained molecular dynamics with explicit solvent using the previously published restraints, along with selected data from the literature are used to check and modify existing parameters of the Karplus equations. The parameterizations obtained using glycosidic torsion angles derived from the original solution and recently determined X-ray structures are also compared.     

Determination of the solution structure of the SH3 domain of human p56 Lck tyrosine kinase

Summary The solution structure of the SH3 domain of human p56 Lck tyrosine kinase (Lck-SH3) has been determined by multidimensional heteronuclear NMR spectroscopy. The structure was calculated from a total of 935 experimental restraints comprising 785 distance restraints derived from 1017 assigned NOE cross peaks and 150 dihedral angle restraints derived from 160 vicinal coupling constants. A novel combination of the constant-time ¹H–¹³C NMR correlation experiment recorded with various delays of the constant-time refocusing delays and a fractionally ¹³C-labelled sample was exploited for the stereo-specific assignment of prochiral methyl groups. Additionally, 28 restraints of 14 identified hydrogen bonds were included. A family of 25 conformers was selected to characterize the solution structure. The average root-mean-square deviations of the backbone atoms (N, C, C, O) among the 25 conformers is 0.42 Å for residues 7 to 63. The N- and C-terminal residues, 1 to 6 and 64 to 81, are disordered, while the well-converged residues 7 to 63 correspond to the conserved sequences of other SH3 domains. The topology of the SH3 structure comprises five anti-parallel -strands arranged to form two perpendicular -sheets, which are concave and twisted in the middle part. The overall secondary structure and the backbone conformation of the core -strands are almost identical to the X-ray structure of the fragment containing the SH2-SH3 domains of p56 Lck [Eck et al. (1994) Nature, 368, 764–769]. The X-ray structure of the SH3 domain in the tandem SH2-SH3 fragment is spatially included within the ensemble of the 25 NMR conformers, except for the segment of residues 14 to 18, which makes intermolecular contacts with an adjacent SH2 molecule and the phosphopeptide ligand in the crystal lattice. Local structural differences from other known SH3 domains are also observed, the most prominent of which is the absence in Lck-SH3 of the two additional short -strands in the regions Ser¹⁵ to Glu¹⁷ and Gly²⁵ to Glu²⁷ flanking the so-called RT-Src loop. This loop (residues Glu¹⁷ to Leu²⁴), together with the n-Src loop (residues Gln³⁷ to Ser⁴⁶) confines the ligand interaction site which is formed by a shallow patch of hydrophobic amino acids (His¹⁴, Tyr¹⁶, Trp⁴¹, Phe⁵⁴ and Phe⁵⁹). Both loops are flexible and belong to the most mobile regions of the protein, which is assessed by the heteronuclear ¹⁵N,¹H-NOE values characterizing the degree of internal backbone motions. The aromatic residues of the ligand binding site are arranged such that they form three pockets for interactions with the polyproline ligand.Abbreviations CT constant time - HSQC heteronuclear single-quantum coherence - NOE nuclear Overhauser enhancement - NOESY nuclear Overhauser enhancement spectroscopy - SH2 Src homology domain 2 - SH3 Src homology domain 3     

Validation of the GROMOS force-field parameter set 45A3 against nuclear magnetic resonance data of hen egg lysozyme

The quality of molecular dynamics (MD) simulations of proteins depends critically on the biomolecular force field that is used. Such force fields are defined by force-field parameter sets, which are generally determined and improved through calibration of properties of small molecules against experimental or theoretical data. By application to large molecules such as proteins, a new force-field parameter set can be validated. We report two 3.5 ns molecular dynamics simulations of hen egg white lysozyme in water applying the widely used GROMOS force-field parameter set 43A1 and a new set 45A3. The two MD ensembles are evaluated against NMR spectroscopic data NOE atom–atom distance bounds, ³J_NH and ³J coupling constants, and ¹5N relaxation data. It is shown that the two sets reproduce structural properties about equally well. The 45A3 ensemble fulfills the atom–atom distance bounds derived from NMR spectroscopy slightly less well than the 43A1 ensemble, with most of the NOE distance violations in both ensembles involving residues located in loops or flexible regions of the protein. Convergence patterns are very similar in both simulations atom-positional root-mean-square differences (RMSD) with respect to the X-ray and NMR model structures and NOE inter-proton distances converge within 1.0–1.5 ns while backbone ³J_HN-coupling constants and ¹H– ¹5N order parameters take slightly longer, 1.0–2.0 ns. As expected, side-chain ³J-coupling constants and ¹H– ¹5N order parameters do not reach full convergence for all residues in the time period simulated. This is particularly noticeable for side chains which display rare structural transitions. When comparing each simulation trajectory with an older and a newer set of experimental NOE data on lysozyme, it is found that the newer, larger, set of experimental data agrees as well with each of the simulations. In other words, the experimental data converged towards the theoretical result.     

A novel method for the biosynthesis of deuterated proteins with selective protonation at the aromatic rings of Phe,Tyr and Trp

A novel biosynthetic strategy is described for the preparation of deuterated proteins containing protons at the ring carbons of Phe, Tyr and Trp, using the aromatic amino acid precursor shikimic acid. Specific protonation at aromatic side chains, with complete deuteration at C^/positions was achieved in proteins overexpressed in bacteria grown in shikimate-supplemented D₂O medium. Co-expression of a shikimate transporter in prototrophic bacteria resulted in protonation levels of 62–79%, whereas complete labeling was accomplished using shikimate auxotrophic bacteria. Our labeling protocol permits the measurement of important aromatic side chain derived distance restraints in perdeuterated proteins that could be utilized to enhance the accuracy of NMR structures calculated using low densities of NOEs from methyl selectively protonated samples.     

Overall structure and sugar dynamics of a DNA dodecamer from homo- and heteronuclear dipolar couplings and 31P chemical shift anisotropy

The solution structure of d(CGCGAATTCGCG)₂ has been determined on the basis of an exceptionally large set of residual dipolar couplings. In addition to the heteronuclear ¹³C-¹H and ¹⁵N-¹H and qualitative homonuclear ¹H-¹H dipolar couplings, previously measured in bicelle medium, more than 300 quantitative ¹H-¹H and 22 ³¹P-¹H dipolar restraints were obtained in liquid crystalline Pf1 medium, and 22 ³¹P chemical shift anisotropy restraints. High quality DNA structures can be obtained solely on the basis of these new restraints, and these structures are in close agreement with those calculated previously on the basis of ¹³C-¹H and ¹⁵N-¹H dipolar couplings. In the newly calculated structures, ³¹P-¹H dipolar and ³Jsub H3 P sub couplings and ³¹P CSA data restrain the phosphodiester backbone torsion angles. The final structure represents a quite regular B-form helix with a modest bending of 10°, which is essentially independent of whether or not electrostatic terms are used in the calculation. Combined, the number of homo- and heteronuclear dipolar couplings significantly exceeds the number of degrees of freedom in the system. Results indicate that the dipolar coupling data cannot be fit by a single structure, but are compatible with the presence of rapid equilibria between C2-endo and C3-endo deoxyribose puckers (sugar switching). The C2-H2/H2 dipolar couplings in B-form DNA are particularly sensitive to sugar pucker and yield the largest discrepancies when fit to a single structure. To resolve these discrepancies, we suggest a simplified dipolar coupling analysis that yields N/S equilibria for the ribose sugar puckers, which are in good agreement with previous analyses of NMR J_HH couplings, with a population of the minor C3-endo form higher for pyrimidines than for purines.     

Simultaneous determination of disulphide bridge topology and three-dimensional structure using ambiguous intersulphur distance restraints: Possibilities and limitations

Knowledge of the native disulphide bridge topology allows the introduction of conformational restraints between remote parts of the peptide chain. This information is therefore of great importance for the successful determination of the three-dimensional structure of cysteine-rich proteins by NMR spectroscopy. In this paper we investigate the limitations of using ambiguous intersulphur restraints [Nilges, M. (1995) J. Mol. Biol., 245, 645–660] associated with NMR experimental information to determine the native disulphide bridge pattern. Using these restraints in a simulated annealing protocol we have determined the correct topology of numerous examples, including a protein with seven disulphide bridges (phospholipase A₂) and a protein in which 25% of the total number of residues are cysteines (-conotoxin GIIIB). We have also characterised the behaviour of the method when only limited experimental data is available, and find that the proposed protocol permits disulphide bridge determination even with a small number of restraints (around 5 NOEs – including a long-range restraint – per residue). In addition, we have shown that under these conditions the use of a reduced penalty function allows the identification of misassigned NOE restraints. These results indicate that the use of ambiguous intersulphur distances with the proposed simulated annealing protocol is a general method for the determination of disulphide bridge topology, particularly interesting in the first steps of NMR study of cysteine-rich proteins. Comparison with previously proposed protocols indicates that the presented method is more reliable and the interpretation of results is straightforward.     

Measurement and application of 1H-19F dipolar couplings in the structure determination of 2′-fluorolabeled RNA

Residual dipolar couplings can provide powerful restraints for determination and refinement of the solution structure of macromolecules. The application of these couplings in nucleic acid structure elucidation can have an especially dramatic impact, since they provide long-range restraints, typically absent in NOE and J-coupling measurements. Here we describe sensitive X-filtered-E.COSY-type methods designed to measure both the sign and magnitude of long-range ¹H-¹⁹F dipolar couplings in selectively fluorine labeled RNA oligonucleotides oriented in solution by a liquid crystalline medium. The techniques for measuring ¹H-¹⁹F dipolar couplings are demonstrated on a 21-mer RNA hairpin, which has been specifically labeled with fluorine at the 2-hydroxyl position of three ribose sugars. Experimentally measured ¹H-¹⁹F dipolar couplings for the 2-deoxy-2-fluoro-sugars located in the helical region of the RNA hairpin were found to be in excellent agreement with values predicted using canonical A-form helical geometry, demonstrating that these couplings can provide accurate restraints for the refinement of RNA structures determined by NMR.     

HYPER: A hierarchical algorithm for automatic determination of protein dihedral-angle constraints and stereospecific CβH2 resonance assignments from NMR data

A new computer program, HYPER, has been developed for automated analysis of protein dihedral angle values and CH₂ stereospecific assignments from NMR data. HYPER uses a hierarchical grid-search algorithm to determine allowed values of , , and ₁ dihedral angles and CH₂ stereospecific assignments based on a set of NMR-derived distance and/or scalar-coupling constraints. Dihedral-angle constraints are valuable for restricting conformational space and improving convergence in three-dimensional structure calculations. HYPER computes the set of , , and ₁dihedral angles and CH₂ stereospecific assignments that are consistent with up to nine intraresidue and sequential distance bounds, two pairs of relative distance bounds, thirteen homo- and heteronuclear scalar coupling bounds, and two pairs of relative scalar coupling constant bounds. The program is designed to be very flexible, and provides for simple user modification of Karplus equations and standard polypeptide geometries, allowing it to accommodate recent and future improved calibrations of Karplus curves. The C code has been optimized to execute rapidly (0.3–1.5 CPU-sec residue^–1 using a 5° grid) on Silicon Graphics R8000, R10000 and Intel Pentium CPUs, making it useful for interactive evaluation of inconsistent experimental constraints. The HYPER program has been tested for internal consistency and reliability using both simulated and real protein NMR data sets.     

Structure refinement of flexible proteins using dipolar couplings: Application to the protein p8MTCP1

The present study deals with the relevance of using mobility-averaged dipolar couplings for the structure refinement of flexible proteins. The 68-residue protein p8^MTCP1 has been chosen as model for this study. Its solution state consists mainly of three -helices. The two N-terminal helices are strapped in a well-determined -hairpin, whereas, due to an intrinsic mobility, the position of the third helix is less well defined in the NMR structure. To further characterize the degrees of freedom of this helix, we have measured the dipolar coupling constants in the backbone of p8^MTCP1 in a bicellar medium. We show here that including D _HN ^dip dipolar couplings in the structure calculation protocol improves the structure of the -hairpin but not the positioning of the third helix. This is due to the motional averaging of the dipolar couplings measured in the last helix. Performing two calculations with different force constants for the dipolar restraints highlights the inconstancy of these mobility-averaged dipolar couplings. Alternatively, prior to any structure calculations, comparing the values of the dipolar couplings measured in helix III to values back-calculated from an ideal helix demonstrates that they are atypical for a helix. This can be partly attributed to mobility effects since the inclusion of the ¹⁵N relaxation derived order parameter allows for a better fit.