Simulated annealing with restrained molecular dynamics using a flexible restraint potential: theory and evaluation with simulated NMR constraints.

A new functional representation of NMR-derived distance constraints, the flexible restraint potential, has been implemented in the program CONGEN (Bruccoleri RE, Karplus M, 1987, Biopolymers 26:137-168) for molecular structure generation. In addition, flat-bottomed restraint potentials for representing dihedral angle and vicinal scalar coupling constraints have been introduced into CONGEN. An effective simulated annealing (SA) protocol that combines both weight annealing and temperature annealing is described. Calculations have been performed using ideal simulated NMR constraints, in order to evaluate the use of restrained molecular dynamics (MD) with these target functions as implemented in CONGEN. In this benchmark study, internuclear distance, dihedral angle, and vicinal coupling constant constraints were calculated from the energy-minimized X-ray crystal structure of the 46-amino acid polypeptide crambin (ICRN). Three-dimensional structures of crambin that satisfy these simulated NMR constraints were generated using restrained MD and SA. Polypeptide structures with extended backbone and side-chain conformations were used as starting conformations. Dynamical annealing calculations using extended starting conformations and assignments of initial velocities taken randomly from a Maxwellian distribution were found to adequately sample the conformational space consistent with the constraints. These calculations also show that loosened internuclear constraints can allow molecules to overcome local minima in the search for a global minimum with respect to both the NMR-derived constraints and conformational energy. This protocol and the modified version of the CONGEN program described here are shown to be reliable and robust, and are applicable generally for protein structure determination by dynamical simulated annealing using NMR data.     

Simulated annealing with restrained molecular dynamics using CONGEN: energy refinement of the NMR solution structures of epidermal and type-alpha transforming growth factors.

The new functionality of the program CONGEN (Bruccoleri RE, Karplus M, 1987, Biopolymers 26:137-168; Bassolino-Klimas D et al., 1996, Protein Sci 5:593-603) has been applied for energy refinement of two previously determined solution NMR structures, murine epidermal growth factor (mEGF) and human type-alpha transforming growth factor (hTGF alpha). A summary of considerations used in converting experimental NMR data into distance constraints for CONGEN is presented. A general protocol for simulated annealing with restrained molecular dynamics is applied to generate NMR solution structures using CONGEN together with real experimental NMR data. A total of 730 NMR-derived constraints for mEGF and 424 NMR-derived constraints for hTGF alpha were used in these energy-refinement calculations. Different weighting schemes and starting conformations were studied to check and/or improve the sampling of the low-energy conformational space that is consistent with all constraints. The results demonstrate that loosened (i.e., "relaxed") sets of the EGF and hTGF alpha internuclear distance constraints allow molecules to overcome local minima in the search for a global minimum with respect to both distance restraints and conformational energy. The resulting energy-refined structures of mEGF and hTGF alpha are compared with structures determined previously and with structures of homologous proteins determined by NMR and X-ray crystallography.     

Strategy for supplementing structure calculations using limited data with hydrophobic distance restraints

Introductory biochemistry texts often note that the fold of a protein is completely defined when the dihedral angles phi and psi are known for each amino acid. This assertion was examined with torsion angle dynamics and simulated annealing (TAD/SA) calculations of protein G using only dihedral angle restraints. When all dihedral angles were restrained to within 1 degrees of the values of the X-ray structure, the TAD/SA structures gave a backbone root mean square deviation to the target of 4 A. Factors that contributed to divergence from the correct solution include deviations of peptide bonds from planarity, internal conflicts resulting from the nonuniform energies of different phi, psi combinations, and relaxation to extended conformations in the absence of long-range constraints. Simulations including hydrogen-bond restraints showed that even a few long-range contacts constrain the fold better than a complete set of accurate dihedral restraints. A procedure is described for TAD/SA calculations using hydrogen-bond restraints, idealized dihedral restraints for residues in regular secondary structures, and "hydrophobic distance restraints" derived from the positions of hydrophobic residues in the amino acid sequence. The hydrogen-bond restraints are treated as inviolable, whereas violated hydrophobic restraints are removed following reduction of restraint upper bounds from 2 to 1 times the predicted radius of gyration. The strategy was tested with simulated restraints from X-ray structures of proteins from different fold classes and NMR data for cold shock protein A that included only backbone chemical shifts and hydrogen bonds obtained from a long-range HNCO experiment.     

Docking multiple conformations of a flexible ligand into a protein binding site using NMR restraints

A method is described for docking a large, flexible ligand using intra-ligand conformational restraints from exchange-transferred NOE (etNOE) data. Numerous conformations of the ligand are generated in isolation, and a subset of representative conformations is selected. A crude model of the protein-ligand complex is used as a template for overlaying the selected ligand structures, and each complex is conformationally relaxed by molecular mechanics to optimize the interaction. Finally, the complexes were assessed for structural quality. Alternative approaches are described for the three steps of the method: generation of the initial docking template; selection of a subset of ligand conformations; and conformational sampling of the complex. The template is generated either by manual docking using interactive graphics or by a computational grid-based search of the binding site. A subset of conformations from the total number of peptides calculated in isolation is selected based on either low energy and satisfaction of the etNOE restraints, or a cluster analysis of the full set. To optimize the interactions in the complex, either a restrained Monte Carlo-energy minimization (MCM) protocol or a restrained simulated annealing (SA) protocol were used. This work produced 53 initial complexes of which 8 were assessed in detail. With the etNOE conformational restraints, all of the approaches provide reasonable models. The grid-based approach to generate an initial docking template allows a large volume to be sampled, and as a result, two distinct binding modes were identified for a fifteen-residue peptide binding to an enzyme active site.     

Practical applications of time-averaged restrained molecular dynamics to ligand-receptor systems: FK506 bound to the Q50R,A95H,K98I triple mutant of FKBP-13

Summary The ability of time-averaged restrained molecular dynamics (TARMD) to escape local low-energy conformations and explore conformational space is compared with conventional simulated-annealing methods. Practical suggestions are offered for performing TARMD calculations with ligand-receptor systems, and are illustrated for the complex of the immunosuppressant FK506 bound to Q50R,A95H,K98I triple mutant FKBP-13. The structure of ¹³C-labeled FK506 bound to triple-mutant FKBP-13 was determined using a set of 87 NOE distance restraints derived from HSQC-NOESY experiments. TARMD was found to be superior to conventional simulated-annealing methods, and produced structures that were conformationally similar to FK506 bound to wild-type FKBP-12. The individual and combined effects of varying the NOE restraint force constant, using an explicit model for the protein binding pocket, and starting the calculations from different ligand conformations were explored in detail.Abbreviations DG distance geometry - dmFKBP-12 double-mutant (R42K,H87V) FKBP-12 - FKBP-12 FK506-binding protein (12 kDa) - FKBP-13 FK506-binding protein (13 kDa) - HSQC heteronuclear single-quantum coherence - K_NOE force constant (penalty) for NOE-derived distance restraints - MD molecular dynamics - NOE nuclear Overhauser effect - SA simulated annealing - TARMD molecular dynamics with time-averaged restraints - tmFKBP-13 triple-mutant (Q50R,A95H,K98I) FKBP-13 - wtFKBP-12 wild-type FKBP-12     

Solution conformations of a trimannoside from nuclear magnetic resonance and molecular dynamics simulations

N-linked oligosaccharides often act as ligands for receptor proteins in a variety of cell recognition processes. Knowledge of the solution conformations, as well as protein-bound conformations, of these oligosaccharides is required to understand these important interactions. In this paper we present a model for the solution conformations sampled by a simple trimannoside, methyl 3, 6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside, which contains two of the most commonly found glycosidic linkages in N-linked oligosaccharides. This model was derived from simulated annealing protocols incorporating distance restraints extracted from NOESY spectra along with torsional restraints computed from three-bond (1)H-(13)C coupling constants measured across the glycosidic bonds. The model was refined in light of unrestrained molecular dynamics simulations conducted in the presence of solvent water. The resulting model depicts a molecule undergoing conformational averaging in solution, adopting four major and two minor conformations. The four major conformations arise from a pair of two-state transitions, one each at the alpha(1-->3) and alpha(1-->6) linkages, whereas the minor conformations result from an additional transition of the alpha(1-->6) linkage. Our data also suggest that the alpha(1-->3) transition is fast and changes the molecular shape slightly, whereas the alpha(1-->6) is much slower and alters the molecular shape dramatically.     

Backbone folding of the polypeptide cardiac stimulant anthopleurin-A determined by nuclear magnetic resonance, distance geometry and molecular dynamics

The solution conformation of the cardiac stimulatory sea anemone polypeptide anthopleurin-A has been characterised using distance geometry and restrained molecular dynamics calculations. A set of 253 approximate interproton distance restraints and 14 peptide backbone torsion angle restraints derived from two-dimensional 1H-NMR spectra at 500 MHz were used as input for these calculations. 13 structures generated by either metric matrix or variable target function distance geometry calculations were refined using energy minimisation and restrained molecular dynamics. The resulting structures contain a region of twisted antiparellel beta-sheet to which two separate regions of unordered chain are linked by three disulphide bonds. Two loops, one including Pro-41 and the other encompassing residues 10-18, are poorly defined by the NOE data.     

Evaluation of site-directed spin labeling for characterizing protein-ligand complexes using simulated restraints

Simulation studies have been performed to evaluate the utility of site-directed spin labeling for determining the structures of protein-ligand complexes, given a known protein structure. Two protein-ligand complexes were used as model systems for these studies: a 1.9-A-resolution x-ray structure of a dihydrofolate reductase mutant complexed with methotrexate, and a 1.5-A-resolution x-ray structure of the V-Src tyrosine kinase SH2 domain complexed with a five-residue phosphopeptide. Nitroxide spin labels were modeled at five dihydrofolate reductase residue positions and at four SH2 domain residue positions. For both systems, after energy minimization, conformational ensembles of the spin-labeled residues were generated by simulated annealing while holding the remainder of the protein-ligand complex fixed. Effective distances, simulating those that could be obtained from (1)H-NMR relaxation measurements, were calculated between ligand protons and the spin labels. These were converted to restraints with several different levels of precision. Restrained simulated annealing calculations were then performed with the aim of reproducing target ligand-binding modes. The effects of incorporating a few supplementary short-range (< or =5.0 A) distance restraints were also examined. For the dihydrofolate reductase-methotrexate complex, the ligand-binding mode was reproduced reasonably well using relatively tight spin-label restraints, but methotrexate was poorly localized using loose spin-label restraints. Short-range and spin-label restraints proved to be complementary. For the SH2 domain-phosphopeptide complex without the short-range restraints, the peptide did not localize to the correct depth in the binding groove; nevertheless, the orientation and internal conformation of the peptide was reproduced moderately well. Use of the spin-label restraints in conjunction with the short-range restraints resulted in relatively well defined structural ensembles. These results indicate that restraints derived from site-directed spin labeling can contribute significantly to defining the orientations and conformations of bound ligands. Accurate ligand localization appears to require either a few supplementary short-range distance restraints, or relatively tight spin-label restraints, with at least one spin label positioned so that some of the restraints draw the ligand into the binding pocket in the latter case.     

Solution-state structure by NMR of zinc-substituted rubredoxin from the marine hyperthermophilic archaebacterium Pyrococcus furiosus.

The three-dimensional solution-state structure is reported for the zinc-substituted form of rubredoxin (Rd) from the marine hyperthermophilic archaebacterium Pyrococcus furiosus, an organism that grows optimally at 100 degrees C. Structures were generated with DSPACE by a hybrid distance geometry (DG)-based simulated annealing (SA) approach that employed 403 nuclear Overhauser effect (NOE)-derived interproton distance restraints, including 67 interresidue, 124 sequential (i-j = 1), 75 medium-range (i-j = 2-5), and 137 long-range (i-j > 5) restraints. All lower interproton distance bounds were set at the sum of the van Der Waals radii (1.8 A), and upper bounds of 2.7 A, 3.3 A, and 5.0 A were employed to represent qualitatively observed strong, medium, and weak NOE cross peak intensities, respectively. Twenty-three backbone-backbone, six backbone-sulfur (Cys), two backbone-side chain, and two side chain-side chain hydrogen bond restraints were include for structure refinement, yielding a total of 436 nonbonded restraints, which averages to > 16 restraints per residue. A total of 10 structures generated from random atom positions and 30 structures generated by molecular replacement using the backbone coordinates of Clostridium pasteurianum Rd converged to a common conformation, with the average penalty (= sum of the square of the distance bounds violations; +/- standard deviation) of 0.024 +/- 0.003 A2 and a maximum total penalty of 0.035 A2. Superposition of the backbone atoms (C, C alpha, N) of residues A1-L51 for all 40 structures afforded an average pairwise root mean square (rms) deviation value (+/- SD) of 0.42 +/- 0.07 A. Superposition of all heavy atoms for residues A1-L51, including those of structurally undefined external side chains, afforded an average pairwise rms deviation of 0.72 +/- 0.08 A. Qualitative comparison of back-calculated and experimental two-dimensional NOESY spectra indicate that the DG/SA structures are consistent with the experimental spectra. The global folding of P. furiosus Zn(Rd) is remarkably similar to the folding observed by X-ray crystallography for native Rd from the mesophilic organism C. pasteurianum, with the average rms deviation value for backbone atoms of residues A1-L51 of P. furiosus Zn(Rd) superposed with respect to residues K2-V52 of C. pasteurianum Rd of 0.77 +/- 0.06 A. The conformations of aromatic residues that compose the hydrophobic cores of the two proteins are also similar. However, P. furiosus Rd contains several unique structural elements, including at least four additional hydrogen bonds and three potential electrostatic interactions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Determination of the solution structures of domains II and III of protein G from Streptococcus by 1H nuclear magnetic resonance.

We have used 1H nuclear magnetic resonance spectroscopy to determine the solution structures of two small (61 and 64 residue) immunoglobulin G (IgG)-binding domains from protein G, a cell-surface protein from Streptococcus strain G148. The two domains differ in sequence by four amino acid substitutions, and differ in their affinity for some subclasses of IgG. The structure of domain II was determined using a total of 478 distance restraints, 31 phi and 9 chi 1 dihedral angle restraints; that of domain III was determined using a total of 445 distance restraints, 31 phi and 9 chi 1 dihedral angle restraints. A protocol which involved distance geometry, simulated annealing and restrained molecular dynamics was used to determine ensembles of 40 structures consistent with these restraints. The structures are found to consist of an alpha-helix packed against a four-stranded antiparallel-parallel-antiparallel beta-sheet. The structures of the two domains are compared to each other and to the reported structure of a similar domain from a protein G from a different strain of Streptococcus. We conclude that the difference in affinity of domains II and III for IgG is due to local changes in amino acid side-chains, rather than a more extensive change in conformation, suggesting that one or more of the residues which differ between them are directly involved in interaction with IgG.     

Metropolis Monte Carlo calculations of DNA structure using internal coordinates and NMR distance restraints: An alternative method for generating a high-resolution solution structure

Summary A new method, a restrained Monte Carlo (rMC) calculation, is demonstrated for generating high-resolution structures of DNA oligonucleotides in solution from interproton distance restraints and bounds derived from complete relaxation matrix analysis of two-dimensional nuclear Overhauser effect (NOE) spectral peak intensities. As in the case of restrained molecular dynamics (rMD) refinement of structures, the experimental distance restraints and bounds are incorporated as a pseudo-energy term (or penalty function) into the mathematical expression for the molecular energy. However, the use of generalized helical parameters, rather than Cartesian coordinates, to define DNA conformation increases efficiency by decreasing by an order of magnitude the number of parameters needed to describe a conformation and by simplifying the potential energy profile. The Metropolis Monte Carlo method is employed to simulate an annealing process. The rMC method was applied to experimental 2D NOE data from the octamer duplex d(GTA-TAATG)·d(CATTATAC). Using starting structures from different locations in conformational space (e.g. A-DNA and B-DNA), the rMC calculations readily converged, with a root-mean-square deviation (RMSD) of <0.3 Å between structures generated using different protocols and starting structures. Theoretical 2D NOE peak intensities were calculated for the rMC-generated structures using the complete relaxation matrix program CORMA, enabling a comparison with experimental intensities via residual indices. Simulation of the vicinal proton coupling constants was carried out for the structures generated, enabling a comparison with the experimental deoxyribose ring coupling constants, which were not utilized in the structure determination in the case of the rMC simulations. Agreement with experimental 2D NOE and scalar coupling data was good in all cases. The rMC structures are quite similar to that refined by a traditional restrained MD approach (RMSD<0.5 Å) despite the different force fields used and despite the fact that MD refinement was conducted with additional restraints imposed on the endocyclic torsion angles of deoxyriboses. The computational time required for the rMC and rMD calculations is about the same. A comparison of structural parameters is made and some limitations of both methods are discussed with regard to the average nature of the experimental restraints used in the refinement.Abbreviations MC Monte Carlo - rMC restrained Monte Carlo - MD molecular dynamics - rMD restrained molecular dynamics - DG distance geometry - EM energy minimization - 2D NOE two-dimensional nuclear Overhauser effect - DQF-COSY double-quantum-filtered correlation spectroscopy - RMSD root-mean-square deviation To whom correspondence should be addressed.     

Solution conformational study of nociceptin an(d its 1-13 and 1-11 fragments using circular dichroism and two-dimensional NMR in conjunction with theoretical conformational analysis.

Conformational studies of nociceptin (NC-NH2), its fully active fragment, NC(1-13)-NH2, and two significantly less potent fragments, NC(1-13)-OH and NC(1-11)-OH, were conducted in water and TFE solutions by the employment of circular dichroism, and in DMSO-d6 by 2DNMR spectroscopy in conjunction with theoretical conformational analysis. The conformations of all thepeptides studied were calculated taking two approaches. The first assumes multiconformational equilibrium of the peptide studied, which is characterized by a set of conformations (and their statistical weight values)obtained from a global conformational analysis using three methods: the electrostatically driven Monte-Carlo (EDMC) with the ECEPP/3 force field, the simulated annealing (SA) protocols in the AMBER and CHARMM force fields. The second approach incorporates the interproton distance and dihedral angle constraints into the starting conformation. Calculations were performed using the distance geometry and SA protocol in the CHARMM force field implemented in the X-PLOR program. The CD experiments indicated that for the active peptides, hydrophobic solvents induced a significantly higher (compared with those remaining)content order, probably a helical structure. Unfortunately, as a result of the conformational flexibility of thepeptides, the analysis of conformations obtained with both approaches and different force fields did not alllow the selection of any structural elements of the NC peptides that might be connected with their bioactivity. The only common element found in most conformations of the active peptides was a helical character of fragment 8-13, which allowed the side chains of basic amino acid residues to be exposed to the outside of the molecule and probably to interact with the ORL1 receptor.     

Low resolution structure of interleukin-1 beta in solution derived from 1H-15N heteronuclear three-dimensional nuclear magnetic resonance spectroscopy

A low resolution solution structure of the cytokine interleukin-1 beta, a 153 residue protein of molecular weight 17,400, has been determined on the basis of 446 nuclear Overhauser effect (NOE) derived approximate interproton distance restraints involving solely NH, C alpha H and C beta H protons, supplemented by 90 distance restraints for 45 hydrogen bonds, and 79 phi torsion angle restraints. With the exception of 27 C alpha H-C alpha H NOEs, all the NOEs were assigned from a three-dimensional 1H-1H NOE 15N-1H heteronuclear multiple quantum coherence (HMQC) spectrum. The torsion angle restraints were obtained from accurate 3JHN alpha coupling constants measured from a HMQC-J spectrum, while the hydrogen bonds were derived from a qualitative analysis of the NOE, coupling constant and amide exchange data. A total of 20 simulated annealing (SA) structures was computed using the hybrid distance geometry-dynamical simulated annealing method. The solution structure of IL-1 beta comprises 12 beta-strands arranged in three pseudo-symmetrical topological units (each consisting of 5 anti-parallel beta-strands), joined by turns, short loops and long loops. The core of the structure, which is made up of the 12 beta-strands, together with the turns joining strands I and II, strands VIII and IX and strands X and XI, is well determined with a backbone atomic root-mean-square (r.m.s.) distribution about the mean co-ordinate positions of 1.2(+/- 0.1) A. The loop conformations, on the other hand, are poorly determined by the current data. A comparison of the core of the low resolution solution structure of IL-1 beta with that of the X-ray structure indicates that they are similar, with a backbone atomic r.m.s. difference of only 1.5 A between the co-ordinates of the restrained minimized mean of the SA structures and the X-ray structure.     

Conformation in solution and dynamics of a structurally constrained linear insect kinin pentapeptide analogue

The preferred conformations of the active diuretic insect kinin pentapeptide analogue Phe-Phe-Aib-Trp-Gly-NH2 were studied using nmr spectroscopy and molecular modeling. Structure sets consistent with rotating frame nuclear Overhauser effect spectroscopy distance constraints obtained by restrained simulated annealing in vacuo indicate a predominant population of a type II beta-turn involving the Phe1-Trp4 region. An equilibrium between this type II and a type I beta-turn formed by residues Phe2 and Gly5 was observed in a 5 ns restrained molecular dynamics simulation using the implicit generalized Born solvent accessible surface area (GB/SA) solvation model. When subjected to 500 ps dynamics with explicit water both beta-turn folds were conserved throughout the simulations. The results obtained with implicit and explicit solvation models are compared, and their consistency with the nmr observations is discussed. The behavior of the linear pentapeptide in this study is in agreement with an earlier report on the consensus conformation of the insect kinin active core derived from analysis of cyclic active analogues.     

Determination of the conformation of d(GGAAATTTCC)2 in solution by use of 1H NMR and restrained molecular dynamics

The conformation of the putative bent DNA d(GGAAATTTCC)2 in solution was studied by use of 1H NMR and restrained molecular dynamics. Most of the resonances were assigned sequentially. A total of 182 interproton distance restraints were determined from two-dimensional nuclear Overhauser effect spectra with short mixing times. Torsion angle restraints for each sugar moiety were determined by qualitative analysis of a two-dimensional correlated spectrum. Restrained molecular dynamics was carried out with the interproton distances and torsion angles incorporated into the total energy function of the system in the form of effective potential terms. As initial conformations for restrained molecular dynamics, classical A-DNA and B-DNA were adopted. The root mean square deviation (rmsd) between these two conformations is 5.5 A. The conformations obtained by use of restrained molecular dynamics are very similar to each other, the rmsd being 0.8 A. On the other hand, the conformations obtained by use of molecular dynamics without experimental restraints or restrained energy minimization depended heavily on the initial conformations, and convergence to a similar conformation was not attained. The conformation obtained by use of restrained molecular dynamics exhibits a few remarkable features. The second G residue takes on the BII conformation [Fratini, A. V., Kopka, M. L., Drew, H. R., & Dickerson, R. E. (1982) J. Biol. Chem. 257, 14686-14707] rather than the standard BI conformation. There is discontinuity of the sugar puckering between the eighth T and ninth C. The minor groove of the oligo(dA) tract is rather compressed. As a result, d(GGAAATTTCC)2 is bent.     

Stabilization of an alpha-helical conformation in an isolated hexapeptide inhibitor of calmodulin.

The conformational properties of two hexapeptides, Ac-LWRILW-NH(2) and its D-amino acid counterpart Ac-lwrilw-NH(2), identified as calmodulin inhibitors using mixture-based synthetic combinatorial library approaches, have been characterised by NMR and CD spectroscopy. The peptides fold into an alpha-helical conformation in aqueous solution. The observed short- and medium-range nuclear Overhauser effects were consistent with the formation of an alpha-helical structure and a reasonably well-defined set of structures was obtained by using restraints from the NMR data in simulated annealing calculations. Analysis of glycine-substitution analogues demonstrated that all the amino acids that make up the peptide sequence are important for the stabilization of the alpha-helical conformation. The results suggest that a well-defined set of interactions is indispensable to allow alpha-helix formation in this short hexapeptide.     

High-resolution three-dimensional structure of reduced recombinant human thioredoxin in solution

The solution structure of recombinant human thioredoxin (105 residues) has been determined by nuclear magnetic resonance (NMR) spectroscopy combined with hybrid distance geometry-dynamical simulated annealing calculations. Approximate interproton distance restraints were derived from nuclear Overhauser effect (NOE) measurements. In addition, a large number of stereospecific assignments for beta-methylene protons and torsion angle restraints for phi, psi, and chi 1 were obtained by using a conformational grid search on the basis of the intraresidue and sequential NOE data in conjunction with 3JHN alpha and 3J alpha beta coupling constants. The structure calculations were based on 1983 approximate interproton distance restraints, 52 hydrogen-bonding restraints for 26 hydrogen bonds, and 98 phi, 71 psi, and 72 chi 1 torsion angle restraints. The 33 final simulated annealing structures obtained had an average atomic rms distribution of the individual structures about the mean coordinate positions of 0.40 +/- 0.06 A for the backbone atoms and 0.78 +/- 0.05 A for all atoms. The solution structure of human thioredoxin consists of a five-stranded beta-sheet surrounded by four alpha-helices, with an active site protrusion containing the two redox-active cysteines. The overall structure is similar to the crystal and NMR structures of oxidized [Katti, S. K., LeMaster, D. M., & Eklund, H. (1990) J. Mol. Biol. 212, 167-184] and reduced [Dyson, J. H., Gippert, G. P., Case, D. A., Holmgren, A., & Wright, P. (1990) Biochemistry 29, 4129-4136] Escherichia coli thioredoxin, respectively, despite the moderate 25% amino acid sequence homology. Several differences, however, can be noted. The human alpha 1 helix is a full turn longer than the corresponding helix in E. coli thioredoxin and is characterized by a more regular helical geometry. The helix labeled alpha 3 in human thioredoxin has its counterpart in the 3(10) helix of the E. coli protein and is also longer in the human protein. In contrast to these structural differences, the conformation of the active site loop in both proteins is very similar, reflecting the perfect sequence identity for a stretch of eight amino acid residues around the redox-active cysteines.     

Intrinsic nature of the three-dimensional structure of proteins as determined by distance geometry with good sampling properties

Summary A protocol for distance geometry calculation is shown to have excellent sampling properties in the determination of three-dimensional structures of proteins from nuclear magnetic resonance (NMR) data. This protocol uses a simulated annealing optimization employing mass-weighted molecular dynamics in four-dimensional space (Havel, T.F. (1991) Prog. Biophys. Mol. Biol., 56, 43–78). It attains an extremely large radius of convergence, allowing a random coil conformation to be used as the initial estimate for the succeeding optimization process. Computations are performed with four systems of simulated distance data as tests of the protocol, using an unconstrained l-alanine 30mer and three different types of proteins, bovine pancreatic trypsin inhibitor, the -amylase inhibitor Tendamistat, and the N-terminal domain of the 434-repressor. The test of the unconstrained polypeptide confirms that the sampled conformational space is that of the statistical random coil. In the larger and more complicated systems of the three proteins, the protocol gives complete convergence of the optimization without any trace of initial structure dependence. As a result of an exhaustive conformational sampling by the protocol, the intrinsic nature of the structures generated with distance restraints derived from NMR data has been revealed. When the sampled structures are compared with the corresponding X-ray structures, we find that the averages of the sampled structures always show a certain pattern of discrepancy from the X-ray structure. This discrepancy is due to the short distance nature of the distance restraints, and correlates with the characteristic shape of the protein molecule.Abbreviations r.m.s.d. root-mean-square deviation - MD molecular dynamics - NMR nuclear magnetic resonance - NOE nuclear Overhauser enhancement - BPTI bovine pancreatic trypsin inhibitor     

Solution structure of endothelin-3 determined using NMR spectroscopy.

The aqueous solution structure of the 21-residue vasoactive peptide hormone endothelin-3 has been determined using high-resolution NMR spectroscopy. A total of 177 proton-proton distance measurements and 5 chi 1 dihedral angle constraints derived from NMR spectra were used to calculate the structure using a combination of distance geometry and dynamical simulated annealing calculations. The calculations reveal a highly ordered, compact conformation in which a helical region extending from K9 to C15 lies in close apposition with the C-terminal hexapeptide; this interaction seems to be largely driven by hydrophobic interactions. Structure-activity studies are interpreted in terms of the conformational features of the calculated endothelin-3 structure.     

A proton nuclear magnetic resonance study of the conformation of bovine anaphylatoxin C5a in solution

The solution conformation of bovine anaphylatoxin C5a has been investigated by nuclear magnetic resonance (NMR) spectroscopy. The 1H-NMR spectrum is assigned in a sequential manner using a variety of two-dimensional NMR techniques. A qualitative interpretation of the short range nuclear Overhauser enhancement data involving the NH, C alpha H and C beta H protons suggests that C5a has four helices comprising residues 5-11, 15-25, 33-39 and 46-61, and is composed of a globular head (residues 5-61) and a C-terminal tail. The polypeptide fold was determined by hybrid distance geometry-dynamical simulated annealing calculations on the basis of 203 approximate interproton distance restraints, 22 distance restraints for 11 intrahelical hydrogen bonds (identified on the basis of the pattern of short range NOEs and slowly exchanging backbone amide protons) and restraints for the 3 disulfide bridges. The overall polypeptide fold is similar to that of the sequence related human recombinant anaphylatoxin C5a [(1988) Proteins 3, 139-145].