Helix, sheet, and polyproline II frequencies and strong nearest neighbor effects in a restricted coil library

A central issue in protein folding is the degree to which each residue's backbone conformational preferences stabilize the native state. We have studied the conformational preferences of each amino acid when the amino acid is not constrained to be in a regular secondary structure. In this large but highly restricted coil library, the backbone preferentially adopts dihedral angles consistent with the polyproline II conformation rather than alpha or beta conformations. The preference for the polyproline II conformation is independent of the degree of solvation. In conjunction with a new masking procedure, the frequencies in our coil library accurately recapitulate both helix and sheet frequencies for the amino acids in structured regions, as well as polyproline II propensities. Therefore, structural propensities for alpha-helices and beta-sheets and for polyproline II conformations in unfolded peptides can be rationalized solely by local effects. In addition, these propensities are often strongly affected by both the chemical nature and the conformation of neighboring residues, contrary to the Flory isolated residue hypothesis.     

Empirical evaluation of the influence of side chains on the conformational entropy of the polypeptide backbone

Changes in amino acid side chains have long been recognized to alterthe range and distribution of ?, ψ angles found in the main chain of polypeptides. Altering the range and distribution of ?, ψ angles also alters the conformational entropy of the flexible denatured state and may thus stabilize or destabilize it relative to the comparatively conformationally rigid native state. A database of 12,320 residues from 61 nonhomologous, high resolution crystal structures was examined to determine the ?, ψ conformational preferences of each of the 20 amino acids. These observed distributions in the native state of proteins are assumed to also reflect the distributions found in the denatured state. The distributionswere used to approximate the energy surface for each residue, allowing the calculation of relative conformational entropies for each residue relative to glycine. In the most extreme case, replacement of glycine by proline, conformational entropy changes will stabilize the native state relative to the denatured state by ?0.82 ± 0.08 kcal/mol at 20°C. Surprisingly, alanine is found to be the most ordered residue other than proline. This unexpected result is a result of the high percentage of alanines found in helical conformations. This either indicates that the observed distributions in the native state do not reflect the distributions in the denatured state, or that alanine is much more likely to adopt a helical conformation in the denatured state than residues with longer side chains. Among those residues with ?, ψ angles compatible with helix incorporation the percentage of alanines actually in helices is very similar to other residues. This and the consistent ordering of alanine relative to other residues regardless of secondary structure are evidence that ?, ψ distributions in native states reflect those in the denatured states. © 1995 Wiley-Liss, Inc.     

Helix propagation and N-cap propensities of the amino acids measured in alanine-based peptides in 40 volume percent trifluoroethanol.

The helix propagation and N-cap propensities of the amino acids have been measured in alanine-based peptides in 40 volume percent trifluoroethanol (40% TFE) to determine if this helix-stabilizing solvent uniformly affects all amino acids. The propensities in 40% TFE are compared with revised values of the helix parameters of alanine-based peptides in water. Revision of the propensities in water is the result of redefining the capping statistical weights and evaluating the helix nucleation constant with N-capping explicitly included in the helix-coil model. The propagation propensities of all amino acids increase in 40% TFE relative to water, but the increases are highly variable. In water, all beta-branched and beta-substituted amino acids are helix breakers. In 40% TFE, the propagation propensities of the nonpolar amino acids increase greatly, leaving charged and neutral polar, beta-substituted amino acids as helix breakers. Glycine and proline are strong helix breakers in both solvents. Free energy differences for helix propagation (delta delta G) between alanine and other nonpolar amino acids are twice as large in water as predicted from side-chain conformational entropies, but delta delta G values in 40% TFE are close to those predicted from side-chain entropies. This dependence of delta delta G on the solvent points to a specific role of water in determining the relative helix propensities of the nonpolar amino acids. The N-cap propensities converge toward a common value in 40% TFE, suggesting that differential solvation by water contributes to the diversity of N-cap values shown by the amino acids.     

Side-chain entropy effects on protein secondary structure formation

Loss of conformational entropy is one of the primary factors opposing protein folding. Both the backbone and side-chain of each residue in a protein will have their freedom of motion restricted in the final folded structure. The type of secondary structure of which a residue is part will have a significant impact on how much side-chain entropy is lost. Side-chain conformational entropies have previously been determined for folded proteins, simple models of unfolded proteins, alpha-helices, and a dipeptide model for beta-strands, but not for polyproline II (PII) helices. In this work, we present side-chain conformational estimates for the three regular secondary structure types: alpha-helices, beta-strands, and PII helices. Entropies are estimated from Monte Carlo computer simulations. Beta-strands are modeled as two structures, parallel and antiparallel beta-strands. Our data indicate that restraining a residue to the PII helix or antiparallel beta-strand conformations results in side-chain entropies equal to or higher than those obtained by restraining residues to the parallel beta-strand conformation. Side-chains in the alpha-helix conformation have the lowest side-chain entropies. The observation that extended structures retain the most side-chain entropy suggests that such structures would be entropically favored in unfolded proteins under folding conditions. Our data indicate that the PII helix conformation would be somewhat favored over beta-strand conformations, with antiparallel beta-strand favored over parallel. Notably, our data imply that, under some circumstances, residues may gain side-chain entropy upon folding. Implications of our findings for protein folding and unfolded states are discussed.     

Alpha-helix stabilization by alanine relative to glycine: roles of polar and apolar solvent exposures and of backbone entropy

The energetics of alpha-helix formation are fairly well understood and the helix content of a given amino acid sequence can be calculated with reasonable accuracy from helix-coil transition theories that assign to the different residues specific effects on helix stability. In internal helical positions, alanine is regarded as the most stabilizing residue, whereas glycine, after proline, is the more destabilizing. The difference in stabilization afforded by alanine and glycine has been explained by invoking various physical reasons, including the hydrophobic effect and the entropy of folding. Herein, the contribution of these two effects and that of hydrophilic area burial is evaluated by analyzing Ala and Gly mutants implemented in three helices of apoflavodoxin. These data, combined with available data for similar mutations in other proteins (22 Ala/Gly mutations in alpha-helices have been considered), allow estimation of the difference in backbone entropy between alanine and glycine and evaluation of its contribution and that of apolar and polar area burial to the helical stabilization typically associated to Gly-->Ala substitutions. Alanine consistently stabilizes the helical conformation relative to glycine because it buries more apolar area upon folding and because its backbone entropy is lower. However, the relative contribution of polar area burial (which is shown to be destabilizing) and of backbone entropy critically depends on the approximation used to model the structure of the denatured state. In this respect, the excised-peptide model of the unfolded state, proposed by Creamer and coworkers (1995), predicts a major contribution of polar area burial, which is in good agreement with recent quantitations of the relative enthalpic contribution of Ala and Gly residues to alpha-helix formation.     

Nature and distribution of sites of temperature-sensitive folding mutations in the gene for the P22 tailspike polypeptide chain

Temperature-sensitive folding (tsf) mutations in gene 9 of bacteriophage P22 interfere with the folding and association of the tailspike polypeptide chain at restrictive temperature. We report here the location and amino acid substitutions for 24 independent tsf mutants. The distribution of these and previously identified mutations is distinctly non-random; all of the 32 unambiguous sites of tsf mutations are located in the central 350 residues of the 666 residue tailspike polypeptide chain. No ts mutation has been found among the N-terminal 140 amino acids, and none among the C-terminal 170 amino acids. Since the physiological defect in these mutants is the destabilization of an early intermediate in the folding pathway, the localization of the mutants suggests that the central region of the chain is critical for formation or stabilization of this early intermediate. The majority of amino acids that served as sites for the tsf mutations were hydrophilic residues. Sixty percent of the replacements of these residues represented charge changes. This probably reflects the selection for mutant sites at the mature protein surface where the substitutions can be best tolerated without interfering with function. None of the sites of tsf mutations were at aromatic residues, and only one proline site was found. Substitutions at these residues may cause lethal folding defects which are not recovered as tsf mutants. The local sequences at tsf sites resemble those reported for turns. Structural studies identify beta-sheet as the dominant secondary structure. These mutations may disrupt the formation of conformational features of beta-sheets which are repeated, such as turns, associations between pairs of strands, or sheet/sheet packing interactions. Such a model accounts for the occurrence of tsf mutations with similar defective phenotypes at multiple positions along the chain.     

Effect of the N3 residue on the stability of the alpha-helix

N3 is the third position from the N terminus in the alpha-helix with helical backbone dihedral angles. All 20 amino acids have been placed in the N3 position of a synthetic helical peptide (CH(3)CO-[AAX AAAAKAAAAKAGY]-NH(2)) and the helix content measured by circular dichroism spectroscopy at 273 K. The dependence of peptide helicity on N3 residue identity has been used to determine a free energy scale by analysis with a modified Lifson-Roig helix coil theory that includes a parameter for the N3 energy (n3). The most stabilizing residues at N3 in rank order are Ala, Glu, Met/Ile, Leu, Lys, Ser, Gln, Thr, Tyr, Phe, Asp, His, and Trp. Free energies for the most destabilizing residues (Cys, Gly, Asn, Arg, and Pro) could not be fitted. The results correlate with N1, N2, and helix interior energies and not at all with N-cap preferences. This completes our work on studying the structural and energetic preferences of the amino acids for the N-terminal positions of the alpha-helix. These results can be used to rationally modify protein stability, help design helices, and improve prediction of helix location and stability.     

Amino acid conformational preferences and solvation of polar backbone atoms in peptides and proteins

Amino acids in peptides and proteins display distinct preferences for alpha-helical, beta-strand, and other conformational states. Various physicochemical reasons for these preferences have been suggested: conformational entropy, steric factors, hydrophobic effect, and backbone electrostatics; however, the issue remains controversial. It has been proposed recently that the side-chain-dependent solvent screening of the local and non-local backbone electrostatic interactions primarily determines the preferences not only for the alpha-helical but also for all other main-chain conformational states. Side-chains modulate the electrostatic screening of backbone interactions by excluding the solvent from the vicinity of main-chain polar atoms. The deficiency of this electrostatic screening model of amino acid preferences is that the relationships between the main-chain electrostatics and the amino acid preferences have been demonstrated for a limited set of six non-polar amino acid types in proteins only. Here, these relationships are determined for all amino acid types in tripeptides, dekapeptides, and proteins. The solvation free energies of polar backbone atoms are approximated by the electrostatic contributions calculated by the finite difference Poisson-Boltzmann and the Langevin dipoles methods. The results show that the average solvation free energy of main-chain polar atoms depends strongly on backbone conformation, shape of side-chains, and exposure to solvent. The equilibrium between the low-energy beta-strand conformation of an amino acid (anti-parallel alignment of backbone dipole moments) and the high-energy alpha conformation (parallel alignment of backbone dipole moments) is strongly influenced by the solvation of backbone polar atoms. The free energy cost of reaching the alpha conformation is by approximately 1.5 kcal/mol smaller for residues with short side-chains than it is for the large beta-branched amino acid residues. This free energy difference is comparable to those obtained experimentally by mutation studies and is thus large enough to account for the distinct preferences of amino acid residues. The screening coefficients gamma(local)(r) and gamma(non-local)(r) correlate with the solvation effects for 19 amino acid types with the coefficients between 0.698 to 0.851, depending on the type of calculation and on the set of point atomic charges used. The screening coefficients gamma(local)(r) increase with the level of burial of amino acids in proteins, converging to 1.0 for the completely buried amino acid residues. The backbone solvation free energies of amino acid residues involved in strong hydrogen bonding (for example: in the middle of an alpha-helix) are small. The hydrogen bonded backbone is thus more hydrophobic than the peptide groups in random coil. The alpha-helix forming preference of alanine is attributed to the relatively small free energy cost of reaching the high-energy alpha-helix conformation. These results confirm that the side-chain-dependent solvent screening of the backbone electrostatic interactions is the dominant factor in determining amino acid conformational preferences.     

Random coil structures in bacterial proteins. Relationships of their amino acid compositions to flanking structures and corresponding genic base compositions

In this study we classified regions of random coil into four types: coil between alpha helix and beta strand, coil between beta strand and alpha helix, coil between two alpha helices and coil between two beta strands. This classification may be considered as natural. We used 610 3D structures of proteins collected from the Protein Data Bank from bacteria with low, average and high genomic GC-content. Relatively short regions of coil are not random: certain amino acid residues are more or less frequent in each of the types of coil. Namely, hydrophobic amino acids with branched side chains (Ile, Val and Leu) are rare in coil between two beta strands, unlike some acrophilic amino acids (Asp, Asn and Gly). In contrast, coil between two alpha helices is enriched by Leu. Regions of coil between alpha helix and beta strand are enriched by positively charged amino acids (Arg and Lys), while the usage of residues with side chains possessing hydroxyl group (Ser and Thr) is low in them, in contrast to the regions of coil between beta strand and alpha helix. Regions of coil between beta strand and alpha helix are significantly enriched by Cys residues. The response to the symmetric mutational pressure (AT-pressure or GC-pressure) is also quite different for four types of coil. The most conserved regions of coil are “connecting bridges” between beta strand and alpha helix, since their amino acid content shows less strong dependence on GC-content of genes than amino acid contents of other three types of coil. Possible causes and consequences of the described differences in amino acid content distribution between different types of random coil have been discussed.     

Backbone entropy of loops as a measure of their flexibility: Application to a Ras protein simulated by molecular dynamics

The flexibility of surface loops plays an important role in protein–protein and protein–peptide recognition; it is commonly studied by Molecular Dynamics or Monte Carlo simulations. We propose to measure the relative backbone flexibility of loops by the difference in their backbone conformational entropies, which are calculated here with the local states (LS) method of Meirovitch. Thus, one can compare the entropies of loops of the same protein or, under certain simulation conditions, of different proteins. These loops should be equal in size but can differ in their sequence of amino acids residues. This methodology is applied successfully to three segments of 10 residues of a Ras protein simulated by the stochastic boundary molecular dynamics procedure. For the first time estimates of backbone entropy differences are obtained, and their correlation with B factors is pointed out; for example, the segments which consist of residues 60–65 and 112–117 have average B factors of 67 and 18 Ų, respectively, and entropy difference T ΔS = 5.4 ± 0.1 kcal/mol at T = 300 K. In a large number of recent publications the entropy due to the fast motions (on the ps-ns time scale) of N–H and C–H vectors has been obtained from their order parameter, measured in nuclear magnetic resonance spin relaxation experiments. This enables one to estimate differences in the entropy of protein segments due to folding–unfolding transitions, for example. However, the vectors are assumed to be independent, and the effect of the neglected correlations is unknown; our method is expected to become an important tool for assessing this approximation. The present calculations, obtained with the LS method, suggest that the errors involved in experimental entropy differences might not be large; however, this should be verified in each case. Potential applications of entropy calculations to rational drug design are discussed. Proteins 29:127–140, 1997. © 1997 Wiley-Liss, Inc.     

A stone's throw and its launch window: timing precision and its implications for language and hominid brains

The Chou-Fasman conformational parameters, P, for amino acid residues in proteins are shown to be a linear function of intermolecular force and steric parameters. For α- helix, coil and turn parameters, steric effects are predominant; whereas for β-sheet parameters, intramolecular forces are predominant. Turn and coil parameters show little or no difference in their dependence which is different from that of α-helix and in some ways almost reciprocal. Factors which increase the probability of finding an amino acid residue in an α-helix usually decrease the probability of finding it in coil or turn. Values of P were calculated for several of the less common amino acids.     

Suppression of the statistical coil state during the α ↔ β transition in homopolypeptides

A matrix formulation of the conformational partition function has been used to examine helix ? sheet transitions in homopolyamino acids. α-Helices are weighted by Zimm-Bragg parameters σ and s. Antiparallel β-sheets with tight bends are weighted by the parameters t, δ, and τ, where t is the propagation parameter. In addition, each bend contributes a factor δ, and each residue in the sheet that does not have a partner in the preceding strand contributes a factor τ. The helix can be the dominant conformation in a long chain only if two conditions are satisfied simultaneously: (i) s > 1 , and (ii) either s > t, or σ, δ, and τ are assigned values that inflict a greater penalty on antiparallel sheets than on helices. The maximum amount of coil developed during the helix ? sheet transition is strongly influenced by the size of τ, but it is only weakly dependent on the size of δ. Previously reported optical rotatory dispersion, CD, laser Raman, and nmr studies of thermally induced α ? β transitions in homopolyamino acids, notably poly(L -lysine), demonstrate that little random coil is present. If the random coil content is to remain small during the helix ? sheet transition, τ must be significantly less than unity. A small value for τ means that there is a significant penalty assessed to lysyl residues in an antiparallel sheet that do not have a partner in a preceding strand.     

Thermodynamic consequences of mutations in vernier zone residues of a humanized anti-human epidermal growth factor receptor murine antibody, 528

To investigate the role of Vernier zone residues, which are comprised in the framework regions and underlie the complementarity-determining regions (CDRs) of antibodies, in the specific, high affinity interactions of antibodies with their targets, we focused on the variable domain fragment of murine anti-human epidermal growth factor receptor antibody 528 (m528Fv). Grafting of the CDRs of m528Fv onto a selected framework region of human antibodies, referred to as humanization, reduced the antibody's affinity for its target by a factor of 1/40. The reduction in affinity was due to a substantial reduction in the negative enthalpy change associated with binding. Crystal structures of the ligand-free antibody fragments showed no noteworthy conformational changes due to humanization, and the loop structures of the CDRs of the humanized antibodies were identical to those of the parent antibodies. Several mutants of the CDR-grafted (humanized) variable domain fragment (h528Fv), in which some of the Vernier zone residues in the heavy chain were replaced with the parental murine residues, were constructed and prepared using a bacterial expression system. Thermodynamic analyses of the interactions between the mutants and the soluble extracellular domain of epidermal growth factor receptor showed that several single mutations and a double mutation increased the negative enthalpy and heat capacity changes. Combination of these mutations, however, led to somewhat reduced negative enthalpy and heat capacity changes. The affinity of each mutant for the target was within the range for the wild-type h528Fv, and this similarity was due to enthalpy-entropy compensation. These results suggest that Vernier zone residues make enthalpic contributions to antigen binding and that the regulation of conformational entropy changes upon humanization of murine antibodies must be carefully considered and optimized.     

Stabilization of secondary structure elements by specific combinations of hydrophilic and hydrophobic amino acid residues is more important for proteins encoded by GC-poor genes

Stabilization of secondary structure elements by specific combinations of hydrophobic and hydrophilic amino acids has been studied by the way of analysis of pentapeptide fragments from twelve partial bacterial proteomes. PDB files describing structures of proteins from species with extremely high and low genomic GC-content, as well as with average G + C were included in the study. Amino acid residues in 78,009 pentapeptides from alpha helices, beta strands and coil regions were classified into hydrophobic and hydrophilic ones. The common propensity scale for 32 possible combinations of hydrophobic and hydrophilic amino acid residues in pentapeptide has been created: specific pentapeptides for helix, sheet and coil were described. The usage of pentapeptides preferably forming alpha helices is decreasing in alpha helices of partial bacterial proteomes with the increase of the average genomic GC-content in first and second codon positions. The usage of pentapeptides preferably forming beta strands is increasing in coil regions and in helices of partial bacterial proteomes with the growth of the average genomic GC-content in first and second codon positions. Due to these circumstances the probability of coil-sheet and helix-sheet transitions should be increased in proteins encoded by GC-rich genes making them prone to form amyloid in certain conditions. Possible causes of the described fact that importance of alpha helix and coil stabilization by specific combinations of hydrophobic and hydrophilic amino acids is growing with the decrease of genomic GC-content have been discussed.     

Solution structures in aqueous SDS micelles of two amyloid beta peptides of A beta(1-28) mutated at the alpha-secretase cleavage site (K16E, K16F)

NMRsolution structures are reported for two mutants (K16E, K16F) of the soluble amyloid beta peptide Abeta(1-28). The structural effects of these mutations of a positively charged residue to anionic and hydrophobic residues at the alpha-secretase cleavage site (Lys16-Leu17) were examined in the membrane-simulating solvent aqueous SDS micelles. Overall the three-dimensional structures were similar to that for the native Abeta(1-28) sequence in that they contained an unstructured N-terminus and a helical C-terminus. These structural elements are similar to those seen in the corresponding regions of full-length Abeta peptides Abeta(1-40) and Abeta(1-42), showing that the shorter peptides are valid model systems. The K16E mutation, which might be expected to stabilize the macrodipole of the helix, slightly increased the helix length (residues 13-24) relative to the K16F mutation, which shortened the helix to between residues 16 and 24. The observed sequence-dependent control over conformation in this region provides an insight into possible conformational switching roles of mutations in the amyloid precursor protein from which Abeta peptides are derived. In addition, if conformational transitions from helix to random coil to sheet precede aggregation of Abeta peptides in vivo, as they do in vitro, the conformation-inducing effects of mutations at Lys16 may also influence aggregation and fibril formation.     

The HIV-1 integrase α4-helix involved in LTR-DNA recognition is also a highly antigenic peptide element

Monoclonal antibodies (MAbas) constitute remarkable tools to analyze the relationship between the structure and the function of a protein. By immunizing a mouse with a 29mer peptide (K159) formed by residues 147 to 175 of the HIV-1 integrase (IN), we obtained a monoclonal antibody (MAba4) recognizing an epitope lying in the N-terminal portion of K159 (residues 147-166 of IN). The boundaries of the epitope were determined in ELISA assays using peptide truncation and amino acid substitutions. The epitope in K159 or as a free peptide (pep-a4) was mostly a random coil in solution, while in the CCD (catalytic core domain) crystal, the homologous segment displayed an amphipathic helix structure (α4-helix) at the protein surface. Despite this conformational difference, a strong antigenic crossreactivity was observed between pep-a4 and the protein segment, as well as K156, a stabilized analogue of pep-a4 constrained into helix by seven helicogenic mutations, most of them involving hydrophobic residues. We concluded that the epitope is freely accessible to the antibody inside the protein and that its recognition by the antibody is not influenced by the conformation of its backbone and the chemistry of amino acids submitted to helicogenic mutations. In contrast, the AA →Glu mutations of the hydrophilic residues Gln148, Lys156 and Lys159, known for their interactions with LTRs (long terminal repeats) and inhibitors (5CITEP, for instance), significantly impaired the binding of K156 to the antibody. Moreover, we found that in competition ELISAs, the processed and unprocessed LTR oligonucleotides interfered with the binding of MAba4 to IN and K156, confirming that the IN α4-helix uses common residues to interact with the DNA target and the MAba4 antibody. This also explains why, in our standard in vitro concerted integration assays, MAba4 strongly impaired the IN enzymatic activity.     

Analysis of secondary structural and physicochemical changes in protein–protein complexes

Conformation switching in protein–protein complexes is considered important for the molecular recognition process. Overall analysis of 123 protein–protein complexes in a benchmark data-set showed that 6.8% of residues switched over their secondary structure conformation upon complex formation. Amino acid residue-wise preference for conformation change has been analyzed in binding and non-binding site residues separately. In this analysis, residues such as Ser, Leu, Glu, and Lys had higher frequency of secondary structural conformation change. The change of helix to coil and sheet to coil conformation and vice versa has been observed frequently, whereas the conformation change of helix to extended sheet occurred rarely in the studied complexes. Influence of conformation change toward the N and C terminal on either side of the binding site residues has been analyzed. Further, analysis on φ and ψ angle variation, conservation, stability, and solvent accessibility have been performed on binding site residues. Knowledge obtained from the present study could be effectively employed in the protein–protein modeling and docking studies.     

Analysis of the conformations corresponding to hexapeptide and large sequences characterized by continuous single amino acid repeats in proteins

The analysis of conformations corresponding to continuous amino acid repeat peptides (CARPs) comprising six or more residues in proteins of known three-dimensional structure revealed that alanine, glycine, glutamic acid, proline, valine, histidine, aspartic acid, glutamine and lysine were associated as repeating amino acid residues. Alanine, glycine and histidine CARPs were most common, although the histidine hexapeptide and large CARPs mainly correspond to affinity tags and are not part of the native protein sequence. The Ala and Glu CARPs were observed either as part of helix, or coil or a combination of these conformations. The octapeptide Ala CARP in six-hairpin glycosidases was observed as part of strand and coil conformation. The Gly and Pro CARPs were mainly associated with coil conformation. Majority of the coil regions in CARPs contained beta and gamma-turn structural motifs. The conformations of the Asp, Glu and Lys hexapeptide or larger CARPs were not defined in the corresponding protein three-dimensional structures analyzed. The longest CARP of known conformation was observed for alanine as a decapeptide in a lysozyme-like protein that corresponds to helix. A feature of CARPs is that a majority are exposed to solvent with accessible surface area greater than 200 ?(2) units in the protein three-dimensional structure.     

Sequence and structural features of binding site residues in protein-protein complexes: comparison with protein-nucleic acid complexes

Background

Protein-protein interactions are important for several cellular processes. Understanding the mechanism of protein-protein recognition and predicting the binding sites in protein-protein complexes are long standing goals in molecular and computational biology.

Methods

We have developed an energy based approach for identifying the binding site residues in protein–protein complexes. The binding site residues have been analyzed with sequence and structure based parameters such as binding propensity, neighboring residues in the vicinity of binding sites, conservation score and conformational switching.

Results

We observed that the binding propensities of amino acid residues are specific for protein-protein complexes. Further, typical dipeptides and tripeptides showed high preference for binding, which is unique to protein-protein complexes. Most of the binding site residues are highly conserved among homologous sequences. Our analysis showed that 7% of residues changed their conformations upon protein-protein complex formation and it is 9.2% and 6.6% in the binding and non-binding sites, respectively. Specifically, the residues Glu, Lys, Leu and Ser changed their conformation from coil to helix/strand and from helix to coil/strand. Leu, Ser, Thr and Val prefer to change their conformation from strand to coil/helix.

Conclusions

The results obtained in this study will be helpful for understanding and predicting the binding sites in protein-protein complexes.

     

Conserved gating hinge in ligand- and voltage-dependent K+ channels

Ion channels open and close their pore in a process called gating. On the basis of crystal structures of two voltage-independent K(+) channels, KcsA and MthK, a conformational change for gating has been proposed whereby the inner helix bends at a glycine hinge point (gating hinge) to open the pore and straightens to close it. Here we ask if a similar gating hinge conformational change underlies the mechanics of pore opening of two eukaryotic voltage-dependent K(+) channels, Shaker and BK channels. In the Shaker channel, substitution of the gating hinge glycine with alanine and several other amino acids prevents pore opening, but the ability to open is recovered if a secondary glycine is introduced at an adjacent position. A proline at the gating hinge favors the open state of the Shaker channel as if by preventing inner helix straightening. In BK channels, which have two adjacent glycine residues, opening is significantly hindered in a graded manner with single and double mutations to alanine. These results suggest that K(+) channels, whether ligand- or voltage-dependent, open when the inner helix bends at a conserved glycine gating hinge.