The interrelationships of side-chain and main-chain conformations in proteins

The accurate determination of a large number of protein structures by X-ray crystallography makes it possible to conduct a reliable statistical analysis of the distribution of the main-chain and side-chain conformational angles, how these are dependent on residue type, adjacent residue in the sequence, secondary structure, residue-residue interactions and location at the polypeptide chain termini. The interrelationship between the main-chain (phi, psi) and side-chain (chi 1) torsion angles leads to a classification of amino acid residues that simplify the folding alphabet considerably and can be a guide to the design of new proteins or mutational studies. Analyses of residues occurring with disallowed main-chain conformation or with multiple conformations shed some light on why some residues are less favoured in thermophiles.     

Electronic and vibrational circular dichroism of aromatic amino acids by density functional theory

Structures of model compounds mimicking aromatic amino acid residues in proteins are optimized by density functional theory (DFT), assuming that the main-chain conformation was a random coil. Excitation energies and dipole and rotational strengths for the optimized structures were calculated based on time-dependent DFT (TD-DFT). The electronic circular dichroism (ECD) bands of the models were significantly affected by side-chain conformations. Hydration models of the aromatic residues were also subjected to TD-DFT calculations, and the ECD bands of these models were found to be highly perturbed by the hydration of the main-chain amide groups. In addition to calculating the random-coil conformation, we also performed TD-DFT calculations of the aromatic residue models, assuming that the main-chain conformation was an alpha-helix or beta-strand. As expected, the overall feature of the ECD bands was also perturbed by the main-chain conformations. Moreover, vibrational circular dichroism (VCD) spectra of the hydration models in a random-coil structure were simulated by DFT, which showed that the VCD spectra are more sensitive to the side-chain conformations than the ECD spectra. The present results show that analyses combining ECD and VCD spectroscopy and using DFT calculations can elucidate the main- and side-chain conformations of aromatic residues in proteins.     

Solution conformation of cytochrome c-551 from Pseudomonas stutzeri ZoBell determined by NMR.

1H NMR spectroscopy and solution structure computations have been used to examine ferrocytochrome c-551 from Pseudomonas stutzeri ZoBell (ATCC 14405). Resonance assignments are proposed for all main-chain and most side-chain protons. Stereospecific assignments were also made for some of the beta-methylene protons and valine methyl protons. Distance constraints were determined based upon nuclear Overhauser enhancements between pairs of protons. Dihedral angle constraints were determined from estimates of scalar coupling constants and intra-residue NOEs. Twenty structures were calculated by distance geometry and refined by energy minimization and simulated annealing on the basis of 1012 interproton distance and 74 torsion angle constraints. Both the main-chain and side-chain atoms are well defined except for two terminal residues, and some side-chain atoms located on the molecular surface. The average root mean squared deviation in the position for equivalent atoms between the 20 individual structures and the mean structure obtained by averaging their coordinates is 0.56 +/- 0.10 A for the main-chain atoms, and 0.95 +/- 0.09 A for all nonhydrogen atoms of residue 3 to 80 plus the heme group. The average structure was compared with an analogous protein, cytochrome c-551 from pseudomonas stutzeri. The main-chain folding patterns are very consistent, but there are some differences, some of which can be attributed to the loss of normally conserved aromatic residues in the ZoBell c-551.     

Analysis of the relationship between side-chain conformation and secondary structure in globular proteins

The relationship between the preferred side-chain dihedral angles and the secondary structure of a residue was examined. The structures of 61 proteins solved to a resolution of 2.0 A (1 A = 0.1 nm) or better were analysed using a relational database to store the information. The strongest feature observed was that the chi 1 distribution for most side-chains in an alpha-helix showed an absence of the g- conformation and a shift towards the t conformation when compared to the non-alpha/beta structures. The exceptions to this tendency were for short polar side-chains that form hydrogen bonds with the main-chain which prefer g+. Shifts in the chi 1 preferences for residues in the beta-sheet were observed. Other side-chain dihedral angles (chi 2, chi 3, chi 4) were found to be influenced by the main-chain. This paper presents more accurate distributions for the side-chain dihedral angles which were obtained from the increased number of proteins determined to high resolution. The means and standard deviations for chi 1 and chi 2 angles are presented for all residues according to the secondary structure of the main-chain. The means and standard deviations are given for the most popular conformations for side-chains in which chi 3 and chi 4 rotations affect the position of C atoms.     

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.     

Deterministic features of side-chain main-chain hydrogen bonds in globular protein structures

A total of 19 835 polar residues from a data set of 250 non-homologous and highly resolved protein crystal structures were used to identify side-chain main-chain (SC-MC) hydrogen bonds. The ratio of the number of SC-MC hydrogen bonds to the total number of polar residues is close to 1:2, indicating the ubiquitous nature of such hydrogen bonds. Close to 56% of the SC-MC hydrogen bonds are local involving side-chain acceptor/donor ('i') and a main-chain donor/acceptor within the window i-5 to i+5. These short-range hydrogen bonds form well defined conformational motifs characterized by specific combinations of backbone and side-chain torsion angles. (a) The Ser/Thr residues show the greatest preference in forming intra-helical hydrogen bonds between the atoms O(gamma)(i) and O(i-4). More than half the examples of such hydrogen bonds are found at the middle of alpha-helices rather than at their ends. The most favoured motif of these examples is alpha(R)alpha(R)alpha(R)alpha(R)(g(-)). (b) These residues also show great preference to form hydrogen bonds between O(gamma)(i) and O(i-3), which are closely related to the previous type and though intra-helical, these hydrogen bonds are more often found at the C-termini of helices than at the middle. The motif represented by alpha(R)alpha(R)alpha(R)alpha(R)(g(+)) is most preferred in these cases. (c) The Ser, Thr and Glu are the most frequently found residues participating in intra-residue hydrogen bonds (between the side-chain and main-chain of the same residue) which are characterized by specific motifs of the form beta(g(+)) for Ser/Thr residues and alpha(R)(g(-)g(+)t) for Glu/Gln. (d) The side-chain acceptor atoms of Asn/Asp and Ser/Thr residues show high preference to form hydrogen bonds with acceptors two residues ahead in the chain, which are characterized by the motifs beta (tt')alphaR and beta(t)alpha(R), respectively. These hydrogen bonded segments, referred to as Asx turns, are known to provide stability to type I and type I' beta-turns. (e) Ser/Thr residues often form a combination of SC-MC hydrogen bonds, with the side-chain donor hydrogen bonded to the carbonyl oxygen of its own peptide backbone and the side-chain acceptor hydrogen bonded to an amide hydrogen three residues ahead in the sequence. Such motifs are quite often seen at the beginning of alpha-helices, which are characterized by the beta(g(+))alpha(R)alpha(R) motif. A remarkable majority of all these hydrogen bonds are buried from the protein surface, away from the surrounding solvent. This strongly indicates the possibility of side-chains playing the role of the backbone, in the protein interiors, to satisfy the potential hydrogen bonding sites and maintaining the network of hydrogen bonds which is crucial to the structure of the protein.     

Solution structure of switch Arc,a mutant with 3(10) helices replacing a wild-type beta-ribbon

Adjacent N11L and L12N mutations in the antiparallel beta-ribbon of Arc repressor result in dramatic changes in local structure in which each beta-strand is replaced by a right-handed helix. The full solution structure of this "switch" Arc mutant shows that irregular 3(10) helices compose the new secondary structure. This structural metamorphosis conserves the number of main-chain and side-chain to main-chain hydrogen bonds and the number of fully buried core residues. Apart from a slight widening of the interhelical angle between alpha-helices A and B and changes in side-chain conformation of a few core residues in Arc, no large-scale structural adjustments in the remainder of the protein are necessary to accommodate the ribbon-to-helix change. Nevertheless, some changes in hydrogen-exchange rates are observed, even in regions that have very similar structures in the two proteins. The surface of switch Arc is packed poorly compared to wild-type, leading to approximately 1000A(2) of additional solvent-accessible surface area, and the N termini of the 3(10) helices make unfavorable head-to-head electrostatic interactions. These structural features account for the positive m value and salt dependence of the ribbon-to-helix transition in Arc-N11L, a variant that can adopt either the mutant or wild-type structures. The tertiary fold is capped in different ways in switch and wild-type Arc, showing how stepwise evolutionary transformations can arise through small changes in amino acid sequence.     

Cis peptide bonds in proteins: residues involved, their conformations, interactions and locations.

An analysis of a non-redundant set of protein structures from the Brookhaven Protein Data Bank has been carried out to find out the residue preference, local conformation, hydrogen bonding and other stabilizing interactions involving cis peptide bonds. This has led to a reclassification of turns mediated by cis peptides, and their average geometrical parameters have been evaluated. The interdependence of the side and main-chain torsion angles of proline rings provided an explanation why such rings in cis peptides are found to have the DOWN puckering. A comparison of cis peptides containing proline and non-proline residues show differences in conformation, location in the secondary structure and in relation to the centre of the molecule, and relative accessibilities of residues. Relevance of the results in mutation studies and the cis-trans isomerization during protein folding is discussed.     

Probing multiple effects on 15N, 13C alpha, 13C beta,and 13C' chemical shifts in peptides using density functional theory

We have used density functional calculations on model peptides to study conformational effects on (15)N, (13)C alpha, (13)C beta, and (13)C' chemical shifts, associated with hydrogen bonding, backbone conformation, and side-chain orientation. The results show a significant dependence on the backbone torsion angles of the nearest three residues. Contributions to (15)N chemical shifts from hydrogen bonding (up to 8 ppm), backbone conformation (up to 13 ppm), side-chain orientation and neighborhood residue effects (up to 22 ppm) are significant, and a unified theory will be required to account for their behavior in proteins. In contrast to this, the dependence on sequence and hydrogen bonding is much less for (13)C alpha and (13)C beta chemical shifts (<0.5 ppm), and moderate for carbonyl carbon shifts (<2 ppm). The effects of side-chain orientation are mainly limited to the residue itself for both nitrogen and carbon, but the chi(1) effect is also significant for the nitrogen shift of the following residue and for the (13)C' shift of the preceding residue. The calculated results are used, in conjunction with an additive model of chemical shift contributions, to create an algorithm for prediction of (15)N and (13)C shifts in proteins from their structure; this includes a model to extrapolate results to regions of torsion angle space that have not been explicitly studied by density functional theory (DFT) calculations. Crystal structures of 20 proteins with measured shifts have been used to test the prediction scheme. Root mean square deviations between calculated and experimental shifts 2.71, 1.22, 1.31, and 1.28 ppm for N, C alpha, C beta, and C', respectively. This prediction algorithm should be helpful in NMR assignment, crystal and solution structure comparison, and structure refinement.     

Analysis of N-terminal capping using carbonyl-carbon chemical shift measurements

The model peptide XAAAAEAAARAAAARamide is used to examine the contributions of an N-terminal capping interaction to the conformation and stability of a helical ensemble. The reference peptide has an alanine residue at position X while the capping peptide has a serine residue at this position. The helical ensemble was characterized using circular dichroism measurements and carbonyl-carbon chemical shift measurements of selectively enriched residues. The distribution of helicity within the ensemble of the reference peptide at pH 11 and 0°C appears symmetrical, having a uniform central helix and frayed ends. This distribution is truncated at pH 6 by the repulsive electrostatic interaction between the positively charged α-amino group and the positively charged end of the helical macrodipole. The capping peptide forms a side-chain/main-chain hydrogen bond involving the serine residue and amide of alanine 4. The presence of this hydrogen bond generates a unique motif in the chemical shift profile of its helical ensemble. The conformational stabilization contributed by this hydrogen bond, although cooperatively distributed throughout the helical ensemble, is preferentially focused within the first helical turn. The stabilization provided by this hydrogen bond is able to offset the truncation of the helical ensemble generated by the repulsive electrostatic interaction observed at pH 6. Proteins 33:167–176, 1998. © 1998 Wiley-Liss, Inc.     

Investigation of the solution conformation of cytochrome c-551 from Pseudomonas stutzeri.

1H NMR spectroscopy and solution structure computations have been used to examine ferrocytochrome c-551 from Pseudomonas stutzeri (ATCC 17588). Resonance assignments are proposed for all main-chain and most side-chain protons. Distance constraints were determined on the basis of nuclear Overhauser enhancements between pairs of protons. Dihedral angle constraints were determined from estimates of scaler coupling constants. Twenty-four structures were calculated by distance geometry and refined by energy minimization and simulated annealing on the basis of 1033 interproton distance and 57 torsion angle constraints. Both the main-chain and side-chain atoms are well defined except for a loop region around residues 34-40, the first two residues at the N-terminus and the last two at the C-terminus, and some side chains located on the molecular surface. The average root mean squared deviation in position for equivalent atoms between the 24 individual structures and the mean structure obtained by averaging their coordinates is 0.54 +/- 0.08 A for the main-chain atoms and 0.97 +/- 0.09 A for all non-hydrogen atoms of residues 3-80 plus the heme group. These structures were compared to the X-ray crystallographic structure of an analogous protein, cytochrome c-551 from Pseudomonas aeruginosa [Matsuura, Takano, & Dickerson (1982) J. Mol. Biol. 156, 389-409). The main-chain folding patterns are very consistent, but there are some differences. The largest difference is in a surface loop segment from residues 34 to 40.     

Molecular dynamics simulations of polypeptide conformations in water: A comparison of alpha, beta, and poly(pro)II conformations.

A significant fraction of the so-called "random coil" residues in globular proteins exists in the left-handed poly(Pro)II conformation. In order to compare the behavior of this secondary structure with that of the other regular secondary structures, molecular dynamics simulations, with the GROMOS suite of programs, of an alanine octapeptide in water, in alpha-helix, beta-strand, and left-handed poly(Pro)II conformations, have been performed. Our results indicate a limited flexibility for the alpha-helix conformation and a relatively larger flexibility for the beta-strand and poly(Pro)II conformations. The behavior of oligopeptides with a starting configuration of beta-strand and poly(Pro)II conformations, both lacking interchain hydrogen bonds, were similar. The (phi, psi) angles reflect a continuum of structures including both beta and P(II) conformations, but with a preference for local P(II) regions. Differences in the network of water molecules involved in hydrogen bonding with the backbone of the polypeptide were observed in local regions of beta and P(II) conformations. Such water bridges help stabilize the P(II) conformation relative to the beta conformation. Proteins 1999;36:400-406.     

Conformational similarity indices between different residues in proteins and alpha-helix propensities

Various amino acid similarity matrices have been derived using data on physicochemical properties and molecular evolution. Conformational similarity indices, CS(XX'), between different residues are computed here using the distribution of the main-chain and side-chain torsion angles and the values have been used to cluster amino acids in proteins. A subset of these parameters, CS(AX') indicates the extent of similarity in the main-chain and side-chain conformations (phi,psi and chi1) of different residues (X) with Ala (A) and is found to have strong correlation with alpha-helix propensities. However, no subset of CS(XX') provides any linear relationship with beta-sheet propensities, suggesting that the conformational feature favouring the location of a residue in an alpha-helix is different from the one favouring the beta-sheet. Conformationally similar residues (close CS(AX) values) have similar steric framework of the side-chain (linear/branched, aliphatic/aromatic), irrespective of the polarity or hydrophobicity. Cooperative nucleation of helix may be facile for a contiguous stretch of residues with high overall CS(AX) values.     

CADB: Conformation Angles DataBase of proteins

Conformation Angles DataBase (CADB) provides an online resource to access data on conformation angles (both main-chain and side-chain) of protein structures in two data sets corresponding to 25% and 90% sequence identity between any two proteins, available in the Protein Data Bank. In addition, the database contains the necessary crystallographic parameters. The package has several flexible options and display facilities to visualize the main-chain and side-chain conformation angles for a particular amino acid residue. The package can also be used to study the interrelationship between the main-chain and side-chain conformation angles. A web based JAVA graphics interface has been deployed to display the user interested information on the client machine. The database is being updated at regular intervals and can be accessed over the World Wide Web interface at the following URL: http://144.16.71.148/cadb/.     

Elucidation of primary structure elements controlling early amyloid beta-protein oligomerization

Assembly of monomeric amyloid beta-protein (A beta) into oligomeric structures is an important pathogenetic feature of Alzheimer's disease. The oligomer size distributions of aggregate-free, low molecular weight A beta 40 and A beta 42 can be assessed quantitatively using the technique of photo-induced cross-linking of unmodified proteins. This approach revealed that low molecular weight A beta 40 is a mixture of monomer, dimer, trimer, and tetramer, in rapid equilibrium, whereas low molecular weight A beta 42 preferentially exists as pentamer/hexamer units (paranuclei), which self-associate to form larger oligomers. Here, photo-induced cross-linking of unmodified proteins was used to evaluate systematically the oligomerization of 34 physiologically relevant A beta alloforms, including those containing familial Alzheimer's disease-linked amino acid substitutions, naturally occurring N-terminal truncations, and modifications altering the charge, the hydrophobicity, or the conformation of the peptide. The most important structural feature controlling early oligomerization was the length of the C terminus. Specifically, the side-chain of residue 41 in A beta 42 was important both for effective formation of paranuclei and for self-association of paranuclei into larger oligomers. The side-chain of residue 42, and the C-terminal carboxyl group, affected paranucleus self-association. A beta 40 oligomerization was particularly sensitive to substitutions of Glu22 or Asp23 and to truncation of the N terminus, but not to substitutions of Phe19 or Ala21. A beta 42 oligomerization, in contrast, was largely unaffected by substitutions at positions 22 or 23 or by N-terminal truncations, but was affected significantly by substitutions of Phe19 or Ala21. These results reveal how specific regions and residues control A beta oligomerization and show that these controlling elements differ between A beta 40 and A beta 42.     

Estimates of the loss of main-chain conformational entropy of different residues on protein folding.

The average contribution of conformational entropy for individual amino acid residues towards the free energy of protein folding is not well understood. We have developed empirical scales for the loss of the main-chain (torsion angles, phi and psi) conformational entropy by taking its side-chain into account. The analysis shows that the main-chain component of the total conformational entropy loss for a residue is significant and reflects intrinsic characteristics associated with individual residues. The values have direct correlation with the hydrophobicity values and this has important bearing on the folding process. Proteins 1999;36:332-339.     

Conversion of a beta-strand to an alpha-helix induced by a single-site mutation observed in the crystal structure of Fis mutant Pro26Ala.

The conversion from an alpha-helix to a beta-strand has received extensive attention since this structural change may induce many amyloidogenic proteins to self-assemble into fibrils and cause fatal diseases. Here we report the conversion of a peptide segment from a beta-strand to an alpha-helix by a single-site mutation as observed in the crystal structure of Fis mutant Pro26Ala determined at 2.0 A resolution. Pro26 in Fis occurs at the point where a flexible extended beta-hairpin arm leaves the core structure. Thus it can be classified as a "hinge proline" located at the C-terminal end of the beta2-strand and the N-terminal cap of the A alpha-helix. The replacement of Pro26 to alanine extends the A alpha-helix for two additional turns in one of the dimeric subunits; therefore, the structure of the peptide from residues 22 to 26 is converted from a beta-strand to an alpha-helix. This result confirms the structural importance of the proline residue located at the hinge region and may explain the mutant''s reduced ability to activate Hin-catalyzed DNA inversion. The peptide (residues 20 to 26) in the second monomer subunit presumably retains its beta-strand conformation in the crystal; therefore, this peptide shows a "chameleon-like" character since it can adopt either an alpha-helix or a beta-strand structure in different environments. The structure of Pro26Ala provides an additional example where not only the protein sequence, but also non-local interactions determine the secondary structure of proteins.     

Conformational Similarity Indices Between Different Residues in Proteins and α-Helix Propensities

Abstract

Various amino acid similarity matrices have been derived using data on physicochemical properties and molecular evolution. Conformational similarity indices, CS_XX′, between different residues are computed here using the distribution of the main-chain and side-chain torsion angles and the values have been used to cluster amino acids in proteins. A subset of these parameters, CS_AX′ indicates the extent of similarity in the main-chain and side-chain conformations (φ ψ and χ₁) of different residues (X) with Ala (A) and is found to have strong correlation with α-helix propensities. However, no subset of CS_XX′ provides any linear relationship with β-sheet propensities, suggesting that the conformational feature favouring the location of a residue in an a-helix is different from the one favouring the β-sheet. Conformationally similar residues (close CS_AX values) have similar steric framework of the side-chain (linear/branched, aliphatic/aromatic), irrespective of the polarity or hydrophobicity. Cooperative nucleation of helix may be facile for a contiguous stretch of residues with high overall CS_AX values.     

Peptide T revisited: conformational mimicry of epitopes of anti-HIV proteins.

Peptide T (ASTTTNYT), a fragment corresponding to residues 185-192 of gp120, the coat protein of HIV, is endowed with several biological properties in vitro, notably inhibition of the binding of both isolated gp120 and HIV-1 to the CD4 receptor, and chemotactic activity. Based on previous nuclear magnetic resonance (NMR) studies performed in our laboratory, which were consistent with a regular conformation of the C-terminal pentapeptide, and SAR studies showing that the C-terminal pentapeptide retains most of the biological properties, we designed eight hexapeptides containing in the central part either the TNYT or the TTNY sequence, and charged residues (D/E/R) at the two ends. Conformational analysis based on NMR and torsion angle dynamics showed that all peptides assume folded conformations. albeit with different geometries and stabilities. In particular, peptides carrying an acidic residue at the N-terminus and a basic residue at the C-terminus are characterized by stable helical structures and retain full chemotactic activity. The solution conformation of peptide ETNYTR displays strong structural similarity to the region 19-26 of both bovine pancreatic and bovine seminal ribonuclease, which are endowed with anti-HIV activity. Moreover, the frequent occurrence, in many viral proteins, of TNYT and TTNY, the two core sequences employed in the design of the hexapeptides studied in the present work, hints that the sequence of the C-terminal pentapeptide TTNYT is probably representative of a widespread viral recognition motif.