Side-chain Dihedral Angles Research Articles

Modeling of loops in protein structures.

Comparative protein structure prediction is limited mostly by the errors in alignment and loop modeling. We describe here a new automated modeling technique that significantly improves the accuracy of loop predictions in protein structures. The positions of all nonhydrogen atoms of the loop are optimized in a fixed environment with respect to a pseudo energy function. The energy is a sum of many spatial restraints that include the bond length, bond angle, and improper dihedral angle terms from the CHARMM-22 force field, statistical preferences for the main-chain and side-chain dihedral angles, and statistical preferences for nonbonded atomic contacts that depend on the two atom types, their distance through space, and separation in sequence. The energy function is optimized with the method of conjugate gradients combined with molecular dynamics and simulated annealing. Typically, the predicted loop conformation corresponds to the lowest energy conformation among 500 independent optimizations. Predictions were made for 40 loops of known structure at each length from 1 to 14 residues. The accuracy of loop predictions is evaluated as a function of thoroughness of conformational sampling, loop length, and structural properties of native loops. When accuracy is measured by local superposition of the model on the native loop, 100, 90, and 30% of 4-, 8-, and 12-residue loop predictions, respectively, had <2 A RMSD error for the mainchain N, C(alpha), C, and O atoms; the average accuracies were 0.59 +/- 0.05, 1.16 +/- 0.10, and 2.61 +/- 0.16 A, respectively. To simulate real comparative modeling problems, the method was also evaluated by predicting loops of known structure in only approximately correct environments with errors typical of comparative modeling without misalignment. When the RMSD distortion of the main-chain stem atoms is 2.5 A, the average loop prediction error increased by 180, 25, and 3% for 4-, 8-, and 12-residue loops, respectively. The accuracy of the lowest energy prediction for a given loop can be estimated from the structural variability among a number of low energy predictions. The relative value of the present method is gauged by (1) comparing it with one of the most successful previously described methods, and (2) describing its accuracy in recent blind predictions of protein structure. Finally, it is shown that the average accuracy of prediction is limited primarily by the accuracy of the energy function rather than by the extent of conformational sampling.

Force field and semiempirical MO conformational analysis of dihydropyridine calcium-channel antagonists

Force field and semiempirical MO calculations were used to investigate the conformational features (dihydropyridine ring puckering, inter-ring and side-chain dihedral angles) of a group of 4-aryl substituted dihydropyridine calcium-channel antagonists. The considered compounds were studied both in vacuo and in water (simulated with the Cosmo approach). For derivatives bearing a basic side chain the corresponding protonated structures were also submitted to MO calculations. The investigation highlighted the conformational flexibility of the dihydropyridine derivatives, the Δ H f of the most stable uncharged conformers of each compound lying in a range of 2–7 kcal·mol −1.

NMR studies of the structure and dynamics of peptide E, an endogenous opioid peptide that binds with high affinity to multiple opioid receptor subtypes.

Structural and dynamic properties of opioid peptide E have been examined in an sodium dodecyl sulfate (SDS) micelle. Structural and dynamic studies both indicate that this peptide exhibits greater segmental mobility than typical structured proteins. An nmr structural analysis of adrenal peptide E in SDS micelles indicated the presence of two well-defined beta-turns, one at the N-terminus encompassing residues 3 to 6, and the second in the region between residues 15 and 18. Certain side chain dihedral angles were also remarkably well defined, such as the chi 1 angle of F4, which exhibited a trans configuration. These calculated structures were based on a set of 9.5 restraints per residue. The backbone dynamics of peptide E in SDS micelles were examined through an analysis of 15N-relaxation parameters. An extended model-free analysis was used to interpret the relaxation data. The overall rotational correlation time is 19.7 ns. the average order parameter S2 is 0.66 +/- 0.15. The N-terminal loop region residues including G3 to R6 have an average order parameter of 0.70 +/- 0.23. The average order parameter lies somewhere between that observed for a random coil (e.g., S2 = 0.3) and that of a well-defined tertiary fold (e.g., S2 = 0.86). This suggests that peptide E in SDS micelles adopts a restricted range of conformations rather than a random coil. Based on the helical structure recently obtained for the highly homologous kappa-agonist dynorphin-A(1-17) and the beta-turn in the same region of peptide E, it is reasonable to assume that these two elements of secondary structure reflect different receptor subtype binding geometries. The intermediate order parameters observed for peptide E in an SDS micelle suggest a degree of dynamic mobility that may enable facile interconversion between helical and beta-turn geometries in the N-terminal agonist domain.

C alpha and C beta carbon-13 chemical shifts in proteins from an empirical database.

We have constructed an extensive database of 13C C alpha and C beta chemical shifts in proteins of solution, for proteins of which a high-resolution crystal structure exists, and for which the crystal structure has been shown to be essentially identical to the solution structure. There is no systematic effect of temperature, reference compound, or pH on reported shifts, but there appear to be differences in reported shifts arising from referencing differences of up to 4.2 ppm. The major factor affecting chemical shifts is the backbone geometry, which causes differences of ca. 4 ppm between typical alpha-helix and beta-sheet geometries for C alpha, and of ca. 2 ppm for C beta. The side-chain dihedral angle chi 1 has an effect of up to 0.5 ppm on the C alpha shift, particularly for amino acids with branched side-chains at C beta. Hydrogen bonding to main-chain atoms has an effect of up to 0.9 ppm, which depends on the main-chain conformation. The sequence of the protein and ring-current shifts from aromatic rings have an insignificant effect (except for residues following proline). There are significant differences between different amino acid types in the backbone geometry dependence; the amino acids can be grouped together into five different groups with different phi, psi shielding surfaces. The overall fit of individual residues to a single non-residue-specific surface, incorporating the effects of hydrogen bonding and chi 1 angle, is 0.96 ppm for both C alpha and C beta. The results from this study are broadly similar to those from ab initio studies, but there are some differences which could merit further attention.

Refined solution structure and backbone dynamics of HIV‐1 Nef

The tendency of HIV-1 Nef to form aggregates in solution, particularly at pH values below 8, together with its large fraction of highly mobile residues seriously complicated determination of its three-dimensional structure, both for heteronuclear solution NMR (Grzesiek et al., 1996a, Nat Struct Biol 3:340-345) and for X-ray crystallography (Lee et al., 1996, Cell 85:931-942). Methods used to determine the Nef structure by NMR at pH 8 and 0.6 mM concentration are presented, together with a detailed description of Nef's secondary and tertiary structure. The described techniques have general applicability for the NMR structure determination of proteins that are aggregating and/or have limited stability at low pH values. Extensive chemical shift assignments are reported for backbone and side chain 1H, 13C, and 15N resonances of the HIV-1 Nef deletion mutants NEF delta 2-39, NEF delta 2-39, delta 159-173, and of NEF delta 2-39, delta 159-173 in complex with the SH3 domain of the Hck tyrosine protein kinase. Besides a type II polyproline helix, Nef's structure consists of three alpha-helices, a 3(10) helix, and a five-stranded anti-parallel beta-sheet. The analysis of 15N relaxation parameters of the backbone amide sites reveals that all the secondary structure elements are non-mobile on the picosecond to nanosecond and on the millisecond time scale. A large number of slowly exchanging amide protons provides evidence for the stability of the Nef core even on the time scale of hours. Significant internal motions on the ps to ns time scale are detected for residues 60 to 71 and for residues 149 to 180, which form solvent-exposed loops. The residues of the HIV-1 protease cleavage site (W57/L58) do not exhibit large amplitude motions on the sub-nanosecond time scale, and their side chains insert themselves into a hydrophobic crevice formed between the C-terminus of helix 1 and the N-terminus of helix 2. A refined structure has been determined based on additional constraints for side-chain and backbone dihedral angles derived from a large number of three-bond J-coupling and ROE data.

Conformational Equilibria in Polypeptides. II. Dihedral-Angle Distribution in Antamanide Based on Three-Bond Coupling Information

The use of3Jcoupling information in deriving dihedral-angle restraints for polypeptide-structure determination in the presence of conformational equilibria is illustrated withantamanide,cyclo(–Val1–Pro2– Pro3– Ala4– Phe5– Phe6– Pro7– Pro8– Phe9– Phe10–). The experimental basis comprises accurate three-bond coupling constants as obtained from both homonuclear [C. Griesinger, O.W. Sørensen, and R.R. Ernst,J. Magn. Reson.75,474 (1987)] and heteronuclear [J. M. Schmidt,J. Magn. Reson.124,298 (1997)] exclusive correlation spectroscopy (E.COSY). For the backbone and side-chain dihedral angles in the nonproline residues, φ and χ1, respectively, probability-distribution functions are derived and evaluated on the basis of χ2statistics and significance estimates. Various motional models are considered in the quantitative compilation of molecular-geometry parameters from spin-system parameters. From the3Jcoupling analysis, antamanide is found to possess a very flexible structure which is consistent with the results previously obtained in homonuclear NOE and13C–T1relaxation studies. To fully agree with experiment, rotamer equilibria must be assumed for almost all of the torsions investigated in the peptide.

Solution structure of human neuropeptide Y.

The three-dimensional structure of synthetic human neuropeptide Y in aqueous solution at pH 3.2 and 37 degrees C was determined from two-dimensional 1H NMR data recorded at 600 MHz. A restraint set consisting of 440 interproton distance restraints inferred from NOEs and 11 backbone and 4 side-chain dihedral angle restraints derived from spin-spin coupling constants was used as input for distance geometry calculations on DIANA and simulated annealing and restrained energy minimization in X-PLOR. The final set of 26 structures is well defined in the region of residues 11-36, with a mean pairwise rmsd of 0.51 A for the backbone heavy atoms (N, C alpha and C) and 1.34 A for all heavy atoms. Residues 13-36 form an amphipathic alpha-helix. The N-terminal 10 residues are poorly defined relative to the helical region, although some elements of local structure are apparent. At least one of the three prolines in the N-terminal region co-exists in both cis and trans conformations. An additional set of 24 distances was interpreted as intermolecular distances within a dimer. A combination of distance geometry and restrained simulated annealing yielded a model of the dimer having antiparallel packing of two helical units, whose hydrophobic faces form a well-defined core. Sedimentation equilibrium experiments confirm the observation that neuropeptide Y associates to form dimers and higher aggregates under the conditions of the NMR experiments. Our results therefore support the structural features reported for porcine neuropeptide Y [Cowley, D.J. et al. (1992) Eur. J. Biochem., 205, 1099-1106] rather than the 'aPP' fold described previously for human neuropeptide Y [Darbon, H. et al. (1992) Eur. J. Biochem., 209, 765-771].

Three-dimensional solution structure of mu-conotoxin GIIIB, a specific blocker of skeletal muscle sodium channels.

The three-dimensional solution structure of mu-conotoxin GIIIB, a 22-residue polypeptide from the venom of the piscivorous cone snail Conus geographus, has been determined using 2D 1H NMR spectroscopy. GIIIB binds with high affinity and selectivity to skeletal muscle sodium channels and is a valuable tool for characterizing both the structure and function of these channels. Structural restraints consisting of 289 interproton distances inferred from NOEs and 9 backbone and 5 side chain dihedral angle restraints from spin-spin coupling constants were used as input for simulated annealing calculations and energy minimization in the program X-PLOR. In addition to the 1H NMR derived information, the 13C resonances of GIIIB were assigned at natural abundance, and hydroxyproline C beta and C gamma chemical shifts were used to distinguish between the cis and trans peptide bond conformations. The final set of 20 structures had mean pairwise rms differences over the whole molecule of 1.22 A for the backbone atoms and 2.48 A for all heavy atoms. For the well-defined region encompassing residues 3-21, the corresponding values were 0.74 and 2.54 A, respectively. GIIIB adopts a compact structure consisting of a distorted 310-helix, a small beta-hairpin, a cis-hydroxyproline, and several turns. The molecule is stabilized by three disulfide bonds, two of which connect the helix and the beta-sheet, forming a structural core with similarities to the CS alpha beta motif [Cornet, B., Bonmatin, J.-M., Hetru, C., Hoffmann, J. A., Ptak, M., & Vovelle, F. (1995) Structure 3, 435-448]. This motif is common to several families of small proteins including scorpion toxins and insect defensins. Other structural features of GIIIB include the presence of eight arginine and lysine side chains that project into the solvent in a radial orientation relative to the core of the molecule. These cationic side chains form potential sites of interaction with anionic sites on sodium channels. The global fold is similar to that reported for mu-conotoxin GIIIA, and the structure of GIIIB determined in this study provides the basis for further understanding of the structure-activity relationships of the mu-conotoxins and for their binding to skeletal muscle sodium channels.

Metal search: a computer program that helps design tetrahedral metal-binding sites.

We describe a computer program (Metal Search) that helps design tetrahedrally coordinated metal binding sites in proteins of known structure. The program takes as input the backbone coordinates of a protein and outputs lists of four residues that might form tetrahedral sites if wild-type amino acids were replaced by cysteine or histidine. The program also outputs the side chain dihedral angles of the amino acids and the coordinates of the predicted metal ion. The only function evaluated by Metal Search is the ability of side chains to meet simple geometric criteria for formation of a tetrahedral site, but these criteria are sufficient to produce a manageably small list that can then be evaluated by other means. The program has been used in the introduction of zinc binding sites in the designed four-helix bundle protein alpha 4 and in the B1 domain of streptococcal protein G, and in both cases the tetrahedral coordination of a bound metal ion has been confirmed (Klemba, M., Gardner, K. H., Marino, S., Clarke, N.D., and Regan, L., Nature: Structural Biology 2:368-373, 1995).

Solution Structure of (Cd2+)1-calbindin D9kReveals Details of the Stepwise Structural Changes along the Apo*rarr;(Ca2+)1II*rarr;(Ca2+)2I,IIBinding Pathway

The three-dimensional solution structure of (Cd2+)1-calbindin D9khas determined by distance geometry, restrained molecular dynamics and relaxation matrix calculations using experimental constraints obtained from two-dimensional1H and15N-1H NMR spectroscopy. The final input data consisted of 1055 NOE distance constraints and 71 dihedral angle constraints, corresponding to 15 constraints per residue on average. The resulting ensemble of 24 structures has no distance or dihedral angle constarints consistently violated by more than 0.07 Å and 1.8°, respectively. The structure is characteristic of an EF-hand protein, with two helix-loop-helix calcium binding motifs joined by a flexible linker, and a short anti-parallel β-type interaction between the two ion-binding sites. The four helices are well defined with a root mean square deviation from the mean coordinates of 0.35 Å for the backbone atoms. The structure of the half-saturated cadmium state was compared with the previously determined solution structures of the apo and fully calcium saturated calbindin D9k. The comparisons were aided by introducing the ensemble averaged distance difference matrix as a tool for analyzing differences between two ensembles of structures. Detailed analyses of differences between the three states in backbone and side-chain dihedral angles, hydrogen bonds, interatomic distances, and packing of the hydrophobic core reveal the reorganization of the protein that occurs upon ion binding. Overall, it was found that (Cd2+)1-calbindin D9k, representing the half-saturated calcium state with an ion in site II, is structurally more similar to the fully calcium-saturated state than the apo state. Thus, for the binding sequence apo→(Ca2+)1II→(Ca2+)2I,II, the structural changes occurring upon ion binding are most pronounced for the first binding step, an observation that bears significantly on the molecular basis for cooperative calcium binding in calbindin D9k.

Three-dimensional structure in solution of the polypeptide cardiac stimulant anthopleurin-A.

The three-dimensional structure in aqueous solution of the 49-residue polypeptide anthopleurin-A (AP-A), from the sea anemone Anthopleura xanthogrammica, has been determined from 1H NMR data. A restraint set consisting of 411 interproton distance restraints inferred from NOEs and 19 backbone and 13 side chain dihedral angle restraints from spin-spin coupling constants, as well as 15 lower bound restraints based on the absence of NOEs in the spectra, was used as input for distance geometry calculations in DIANA and simulated annealing and restrained energy minimization in X-PLOR. Stereospecific assignments for 12 beta-methylene pairs were also included. The final set of 20 structures had mean pairwise rms differences over the whole molecule of 2.04 A for the backbone heavy atoms (N, C alpha, and C) and 2.59 A for all heavy atoms. For the well-defined region encompassing residues 2-7 and 17-49, the corresponding values were 0.82 and 1.27 A, respectively. AP-A adopts a compact structure consisting of four short strands of antiparallel beta-sheet (residues 2-4, 20-23, 34-37, and 45-48) connected by three loops. The first loop commences with a type I beta-turn which includes two important Asp residues; this loop is the least well-defined region of the protein, although a beta-turn involving residues 13-16 is observed in nearly half the structures. The loop linking the second and third strands is constrained by the 29-47 disulfide bond and contains two well-defined beta-turns, while the third loop contains the Gly40-Pro41 sequence, which has been identified previously as the site of cis-trans isomerism. The carboxylate group of Asp7 is close to the epsilon-ammonium group of Lys37, suggesting that they may form a salt bridge. A pH titration monitored by 2D NMR supports this by showing that Asp7 has a low pKa. It is proposed that this region of the molecule and the nearby residues Asp9 and His39 form part of the molecular surface which interacts with the mammalian cardiac sodium channel.

Structural studies of the engrailed homeodomain

The structure of the Drosophila engrailed homeodomain has been solved by molecular replacement and refined to an R-factor of 19.7% at a resolution of 2.1 A. This structure offers a high-resolution view of an important family of DNA-binding proteins and allows comparison to the structure of the same protein bound to DNA. The most significant difference between the current structure and that of the 2.8-A engrailed-DNA complex is the close packing of an extended strand against the rest of the protein in the unbound protein. Structural features of the protein not previously noted include a "herringbone" packing of 4 aromatic residues in the core of the protein and an extensive network of salt bridges that covers much of the helix 1-helix 2 surface. Other features that may play a role in stabilizing the native state include the interaction of buried carbonyl oxygen atoms with the edge of Phe 49 and a bias toward statistically preferred side-chain dihedral angles. There is substantial disorder at both ends of the 61 amino acid protein. A 51-amino acid variant of engrailed (residues 6-56) was synthesized and shown by CD and thermal denaturation studies to be structurally and thermodynamically similar to the full-length domain.

Three-dimensional structure in solution of neurotoxin III from the sea anemone Anemonia sulcata.

The three-dimensional structure in aqueous solution of the 27-residue polypeptide neurotoxin Anemonia sulcata toxin III (ATX III) has been determined from 1H NMR data. As ATX III self-associates in the millimolar concentration range, causing a marked concentration dependence for the chemical shifts of several residues [Norton, R. S., Cross, K., Braach-Maksvytis, V., & Wachter, E. (1993) Biochem. J. 293, 545-551], it was necessary to record NOESY spectra over a range of concentrations in order to eliminate any intermolecular interactions from the NOE restraint set. The pairings of the six half-cystine residues were also unknown and had to be determined (as 3-17, 4-11, and 6-22) from preliminary structure calculations performed using both upper bound distance restraints from NOESY data and a substantial number of lower bound restraints inferred from the absence of NOESY cross-peaks. Final structures were determined, using the program X-PLOR, from interproton distance restraints inferred from NOEs, backbone and side chain dihedral angle restraints from spin-spin coupling measurements, and a smaller number of lower bound restraints. Stereospecific assignments for 11 beta-methylene pairs were also included. The final set of 28 structures had an average pairwise RMS difference of 1.32 A over the backbone heavy atoms (N, C alpha, and C) and 2.18 A over all heavy atoms. For the well-defined region encompassing residues 3-22, the corresponding values were 0.62 and 1.28 A, respectively. ATX III adopts a compact structure containing four reverse turns (a distorted type I beta-turn at residues 6-9, a type I beta-turn at residues 8-11, and inverse gamma-turns at residues 12-14 and 15-17) and two other chain reversals, but no regular alpha-helix or beta-sheet. Several of the residues most affected by aggregation are located on the surface of the molecule, forming a hydrophobic patch which may constitute part of the sodium channel binding surface. Possible relationships between the structure of ATX III and those of other sea anemone toxins that interact with the same site on the voltage-gated sodium channel are considered.

Multiple copy sampling in protein loop modeling: Computational efficiency and sensitivity to dihedral angle perturbations

Multiple copy sampling and the bond scaling-relaxation technique are combined to generate 3-dimensional conformations of protein loop segments. The computational efficiency and sensitivity to initial loop copy dispersion are analyzed. The multicopy loop modeling method requires approximately 20-50% of the computational time required by the single-copy method for the various protein segments tested. An analytical formula is proposed to estimate the computational gain prior to carrying out a multicopy simulation. When 7-residue loops within flexible proteins are modeled, each multicopy simulation can sample a set of loop conformations with initial dispersions up to +/- 15 degrees for backbone and +/- 30 degrees for side-chain rotatable dihedral angles. The dispersions are larger for shorter and smaller for longer and/or surface loops. The degree of convergence of loop copies during a simulation can be used to complement commonly used target functions (such as potential energy) for distinguishing between native and misfolded conformations. Furthermore, this convergence also reflects the conformational flexibility of the modeled protein segment. Application to simultaneously building all 6 hypervariable loops of an antibody is discussed.

1H NMR studies on an Asn-linked glycopeptide. GlcNAc-1 C2-N2 bond is rigid in H2O.

The conformation of an Asn-linked glycopeptide in H2O was studied by two-dimensional 1H NMR. Nonexchangeable proton and exchangeable amide (NH) proton resonances were assigned for the hen ovomucoid glycopeptide 1, Ser-Ile-Glu-Phe-Gly-Thr-Asn Ile-Ser-Lys, with pentasaccharide Man alpha 1-3 (Man alpha 1-6)Man beta 1-4GlcNAc beta 1-4GlcNAc beta 1-NH attached to the Asn7 gamma-carboxamide. The pentasaccharide increases the local correlation times of amino acid residues near the N-glycosylation site. Nuclear Overhauser effect (NOE) measurements on 1 and the corresponding Man3-GlcNAc2 pentasaccharide 3 show that the attached peptide does not perturb O-glycoside conformation. Sequential dNN (i, i + 1) NOEs in the Thr6-Ser9 region indicate populations of folded structure near the N-glycosylation site of both glycopeptide 1 and aglycosyl peptide 2. However, the Man3GlcNAc2 pentasaccharide does not dramatically affect the average conformation of either the peptide backbone or the Asn7 side chain. GlcNAc NH protons were studied at pH 3.0; and NOE and 3JNH data were used to constrain the glycopeptide's GlcNAc-1 side chain dihedral angle (tau) (C1-C2-N2-C7(Ac)). The glycopeptide's core GlcNAc-1 C2-N2 side chain bond is not flexible in H2O. A strong GlcNAc-1 NH2-H3 NOE, a medium strength NH2-H1 NOE, and a weak NH2-H2 interaction suggest that GlcNAc-1 has a rigid C2-N2 bond, with tau between 95 and 115 degrees. No evidence was found for intramolecular hydrogen bonds restricting this C2 side chain torsion. It may be that GlcNAc-1's rigid planar N-glycosidic linkage limits the conformational space available to the adjacent C2 acetamido side chain.

Prediction of Protein Side-chain Conformations from Local Three-dimensional Homology Relationships

A method for predicting the conformations of protein side-chains, starting from main-chain co-ordinates alone, is described. The method involves the comparison of the local environment of each residue whose side-chain conformation is to be predicted with a database of local environments for the same residue type constructed from an analysis of high-resolution protein structures. Local environments are described in terms of the residue type and location in space of residues that interact with the side-chain of interest. The best (most three-dimensionally homologous) few matches to each residue are then input to a Monte-Carlo procedure to give a final predicted structure. The method has been tested on a selection of eight proteins, ranging in size from 46 to 323 amino acid residues. The average side-chain atom root-mean-square deviation between the actual and predicted structures is 1·71 Å taken over all residues, and 1·00 Å if restricted to buried residues. Over all residues, an average of 59·8% of all side-chain dihedral angles are predicted within ±30 degrees of the crystal structure values. Considering buried residues only, this rises to 79·6%.

Comparative Protein Modelling by Satisfaction of Spatial Restraints

We describe a comparative protein modelling method designed to find the most probable structure for a sequence given its alignment with related structures. The three dimensional (3D) model is obtained by optimally satisfying spatial restraints derived from the alignment and expressed as probability density functions (pdfs) for the features restrained. For example, the probabilities for main-chain conformations of a modelled residue may be restrained by its residue type, main-chain conformation of an equivalent residue in a related protein, and the local similarity between the two sequences. Several such pdfs are obtained from the correlations between structural features in 17 families of homologous proteins which have been aligned on the basis of their 3D structures. The pdfs restrain Cα-Cα distances, main-chain N-O distances, main-chain and side-chain dihedral angles. A smoothing procedure is used in the derivation of these relationships to minimize the problem of a sparse database. The 3D model of a protein is obtained by optimization of the molecular pdf such that the model violates the input restraints as little as possible. The molecular pdf is derived as a combination of pdfs restraining individual spatial features of the whole molecule. The optimization procedure is a variable target function method that applies the conjugate gradients algorithm to positions of all non hydrogen atoms. The method is automated and is illustrated by the modelling of trypsin from two other serine proteinases.

Backbone-dependent Rotamer Library for Proteins Application to Side-chain Prediction

A backbone-dependent rotamer library for amino acid side-chains is developed and used for constructing protein side-chain conformations from the main-chain co-ordinates. The rotamer library is obtained from 132 protein chains in the Brookhaven Protein Database. A grid of 20° by 20° blocks for the main-chain angles φ,ψ is used in the rotamer library. Significant correlations are found between side-chain dihedral angle probabilities and backbone φ,ψ values. These probabilities are used to place the side-chain on the known backbone in the test application for six proteins for which high-resolution crystal structures are available. A minimization scheme is used to reorient side-chains that conflict with the backbone or other side-chains after the initial placement. The initial placement yields 59% of both χ1 and χ2 values in the correct position (to within 40°) for thermolysin to 81% for crambin. After refinement the values range from 61% (lysozyme) to 89% (crambin). It is evident from the results that a single protein does not adequately test a prediction scheme.The computation time required by the method scales linearly with the number of side-chains. An initial prediction from the library takes only a few seconds of computer time, while the iterative refinement takes on the order of hours. The method is automated and can easily be applied to aid experimental side-chain determinations and homology modeling. The high degree of correlation between backbone and side-chain conformations may introduce a simplification in the protein folding process by reducing the available conformational space.

β-Breakers: An aperiodic secondary structure

We have studied the architecture of parallel β-sheets in proteins and focused on the residues that initiate and terminate the β-strands. These β-breaker residues are at the origin of the kink between the β-strand and the turn that precedes or follows it. β-Breakers can be located automatically using a consensus approach based on algorithmic secondary structure assignment, solvent accessibility and backbone dihedral angles. These β-breakers are conformationally homogeneous with respect to side-chain solvent accessibility and backbone dihedral angle profile. A sequence-structure correlation is noted: a restricted subset of amino acids is observed at these positions. Analysis of homologous protein sequences shows that these residues are more highly conserved than other residues in the loop. We conclude that β-breakers are the structural analogs of the N and C-terminal caps of α-helices. The identification of this aperiodic substructure suggests a strategy for improving secondary structure prediction and may guide site-directed mutagenesis experiments.

Molecular mechanics studies of model polysilanes

A molecular mechanics study of a homologous series of di-substituted polysilane model compounds investigates the effect ofn-alkyl side-chain substituent length on overall polymer conformation. Using the recently released MM3 program to probe backbone and side-chain energetics, the results suggest that an off-trans (160°) backbone structure is optimal in both longer-side chain (i.e., di-n-hexyl) and shorter-side chain (di-ethyl) polysilanes. Also a “chair-like” conformation for side-chain dihedral angles is preferred.