Short peptides as predictors for the structure of polyarginine sequences in disordered proteins

Intrinsically disordered proteins and intrinsically disordered regions are frequently enriched in charged amino acids. Intrinsically disordered regions are regularly involved in important biological processes in which one or more charged residues is the driving force behind a protein-biomolecule interaction. Several lines of experimental and computational evidence suggest that polypeptides and proteins that carry high net charges have a high preference for extended conformations with average end-to-end distances exceeding expectations for self-avoiding random coils. Here, we show that charged arginine residues even in short glycine-capped model peptides (GRRG and GRRRG) significantly affect the conformational propensities of each other when compared with the intrinsic propensities of a mostly unperturbed arginine in the tripeptide GRG. A conformational analysis based on experimentally determined J-coupling constants from heteronuclear NMR spectroscopy and amide I′ band profiles from vibrational spectroscopy reveals that nearest-neighbor interactions stabilize extended β-strand conformations at the expense of polyproline II and turn conformations. The results from molecular dynamics simulations with a CHARMM36m force field and TIP3P water reproduce our results only to a limited extent. The use of the Ramachandran distribution of the central residue of GRRRG in a calculation of end-to-end distances of polyarginines of different length yielded the expected power law behavior. The scaling coefficient of 0.66 suggests that such peptides would be more extended than predicted by a self-avoiding random walk. Our findings thus support in principle theoretical predictions.     

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.     

Disorder and order in unfolded and disordered peptides and proteins: A view derived from tripeptide conformational analysis. I. Tripeptides with long and predominantly hydrophobic side chains

We performed a conformational analysis of the central residues of three tripeptides glycyl‐L ‐isoleucyl‐glycine (GIG), glycyl‐L ‐tyrosyl‐glycine (GYG) and glycyl‐L ‐arginyl‐glycine (GRG) in aqueous solution, based on a global analysis of amide I′ band profiles and NMR J‐coupling constants. The results are compared with recently reported distributions of GVG, GFG and GEG. For GIG and GYG, we found that even though the polyproline II (pPII) fraction is below 0.5, it is still the most populated conformation, whereas GVG and GFG show both a larger β‐strand fraction. For GRG, we observed a clear dominance of pPII over β‐strand, reminiscent of observations for GEG and GKG. This finding indicates that terminal charges on otherwise hydrophobic residue side chains stabilize pPII over β‐strand conformations. For all peptides investigated we found that a variety of compact and turn‐like conformations constitute nearly 20 percent of their conformational distributions. Attempts to analyze our data with a simple two‐state pPII??β model therefore do not yield any satisfactory reproduction of experimental results. A comparison of the obtained GxG ensembles with conformational distributions of GxG segments in truncated coil libraries (helices and sheets omitted) revealed a much larger fraction of type II β_i+2 and type III β like conformations for the latter. Thus, a comparison of conformational distributions of unfolded peptide segments in solution and in coil libraries reveal interesting information on how the interplay between intrinsic propensities of amino acid residues and non‐local interactions in polypeptide chains determine the conformations of loop segments in proteins. Proteins 2013; © 2012 Wiley Periodicals, Inc.     

Anticooperative Nearest-Neighbor Interactions between Residues in Unfolded Peptides and Proteins

Growing evidence suggests that the conformational distributions of amino acid residues in unfolded peptides and proteins depend on the nature of the nearest neighbors. To explore whether the underlying interactions would lead to a breakdown of the isolated pair hypothesis of the classical random coil model, we further analyzed the conformational propensities that were recently obtained for the two guest residues (x,y) of GxyG tetrapeptides. We constructed a statistical thermodynamics model that allows for cooperative as well as for anticooperative interactions between adjacent residues adopting either a polyproline II or a β-strand conformation. Our analysis reveals that the nearest-neighbor interactions between most of the central residues in the investigated GxyG peptides are anticooperative. Interaction Gibbs energies are rather large at high temperatures (350 K), at which point many proteins undergo thermal unfolding. At room temperature, these interaction energies are less pronounced. We used the obtained interaction parameter in a Zimm-Bragg/Ising-type approach to calculate the temperature dependence of the ultraviolet circular dichroism (CD) of the MAX3 peptide, which is predominantly built by KV repeats. The agreement between simulation and experimental data was found to be satisfactory. Finally, we analyzed the temperature dependence of the CD and ³J(H^NH^α) parameters of the amyloid β_1–9 fragment. The results of this analysis and a more qualitative consideration of the temperature dependence of denatured proteins probed by CD spectroscopy further corroborate the dominance of anticooperative nearest-neighbor interactions. Generally, our results show that unfolded peptides—and most likely also proteins—exhibit some similarity with antiferromagnetic systems.     

Disorder and order in unfolded and disordered peptides and proteins: A view derived from tripeptide conformational analysis. II. Tripeptides with short side chains populating asx and β‐type like turn conformations

In the preceding paper, we found that ensembles of tripeptides with long or bulky chains can include up to 20% of various turns. Here, we determine the structural and thermodynamic characteristics of GxG peptides with short polar and/or ionizable central residues (D, N, C), whose conformational distributions exhibit higher than average percentage (>20%) of turn conformations. To probe the side‐chain conformations of these peptides, we determined the ³J(H^α,H^β) coupling constants and derived the population of three rotamers with χ₁‐angles of ?60°, 180° and 60°, which were correlated with residue propensities by DFT‐calculations. For protonated GDG, the rotamer distribution provides additional evidence for asx‐turns. A comparison of vibrational spectra and NMR coupling constants of protonated GDG, ionized GDG, and the protonated aspartic acid dipeptide revealed that side chain protonation increases the pPII content at the expense of turn populations. The charged terminal groups, however, have negligible influence on the conformational properties of the central residue. Like protonated GDG, cationic GCG samples asx‐turns to a significant extent. The temperature dependence of the UVCD spectra and ³J(H^NH^α) constants suggest that the turn populations of GDG and GNG are practically temperature‐independent, indicating enthalpic and entropic stabilization. The temperature‐independent J‐coupling and UVCD spectra of GNG require a three‐state model. Our results indicate that short side chains with hydrogen bonding capability in GxG segments of proteins may serve as hinge regions for establishing compact structures of unfolded proteins and peptides. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

A propensity scale for type II polyproline helices (PPII): aromatic amino acids in proline-rich sequences strongly disfavor PPII due to proline-aromatic interactions

Type II polyproline helices (PPII) are a fundamental secondary structure of proteins, common in globular and nonglobular regions and important in cellular signaling. We developed a propensity scale for PPII using a host-guest system with sequence Ac-GPPXPPGY-NH(2), where X represents any amino acid. We found that proline has the highest PPII propensity, but most other amino acids display significant PPII propensities. The PPII propensity of leucine was the highest of all propensities of non-proline residues. Alanine and residues with linear side chains displayed the next highest PPII propensities. Three classes of residues displayed lower PPII propensities: β-branched amino acids (Thr, Val, and Ile), short amino acids with polar side chains (Asn, protonated Asp, Ser, Thr, and Cys), and aromatic amino acids (Phe, Tyr, and Trp). tert-Leucine particularly disfavored PPII. The basis of the low PPII propensities of aromatic amino acids in this context was significant cis-trans isomerism, with proline-rich peptides containing aromatic residues exhibiting 45-60% cis amide bonds, due to Pro-cis-Pro-aromatic and aromatic-cis-Pro amide bonds.     

Highly Disordered Amyloid-β Monomer Probed by Single-Molecule FRET and MD Simulation

Monomers of amyloid-β (Aβ) protein are known to be disordered, but there is considerable controversy over the existence of residual or transient conformations that can potentially promote oligomerization and fibril formation. We employed single-molecule Förster resonance energy transfer (FRET) spectroscopy with site-specific dye labeling using an unnatural amino acid and molecular dynamics simulations to investigate conformations and dynamics of Aβ isoforms with 40 (Aβ40) and 42 residues (Aβ42). The FRET efficiency distributions of both proteins measured in phosphate-buffered saline at room temperature show a single peak with very similar FRET efficiencies, indicating there is apparently only one state. 2D FRET efficiency-donor lifetime analysis reveals, however, that there is a broad distribution of rapidly interconverting conformations. Using nanosecond fluorescence correlation spectroscopy, we measured the timescale of the fluctuations between these conformations to be ～35 ns, similar to that of disordered proteins. These results suggest that both Aβ40 and Aβ42 populate an ensemble of rapidly reconfiguring unfolded states, with no long-lived conformational state distinguishable from that of the disordered ensemble. To gain molecular-level insights into these observations, we performed molecular dynamics simulations with a force field optimized to describe disordered proteins. We find, as in experiments, that both peptides populate configurations consistent with random polymer chains, with the vast majority of conformations lacking significant secondary structure, giving rise to very similar ensemble-averaged FRET efficiencies.     

Native and nonnative conformational preferences in the urea-unfolded state of barstar

The refolding of barstar from its urea-unfolded state has been studied extensively using various spectroscopic probes and real-time NMR, which provide global and residue-specific information, respectively, about the folding process. Here, a preliminary structural characterization by NMR of barstar in 8 M urea has been carried out at pH 6.5 and 25 degrees C. Complete backbone resonance assignments of the urea-unfolded protein were obtained using the recently developed three-dimensional NMR techniques of HNN and HN(C)N. The conformational propensities of the polypeptide backbone in the presence of 8 M urea have been estimated by examining deviations of secondary chemical shifts from random coil values. For some residues that belong to helices in native barstar, 13C(alpha) and 13CO secondary shifts show positive deviations in the urea-unfolded state, indicating that these residues have propensities toward helical conformations. These residues are, however, juxtaposed by residues that display negative deviations indicative of propensities toward extended conformations. Thus, segments that are helical in native barstar are unlikely to preferentially populate the helical conformation in the unfolded state. Similarly, residues belonging to beta-strands 1 and 2 of native barstar do not appear to show any conformational preferences in the unfolded state. On the other hand, residues belonging to the beta-strand 3 segment show weak nonnative helical conformational preferences in the unfolded state, indicating that this segment may possess a weak preference for populating a helical conformation in the unfolded state.     

Origin of the sequence-dependent polyproline II structure in unfolded peptides

Recent studies have indicated that the unfolded states of polypeptides contain a substantial amount of polyproline type II (P(II)) structure. This energetically and structurally preorganized state may contribute to the reduction of the folding search, as well as to the recognition of intrinsically unstructured proteins and polyproline ligands. Using Monte Carlo simulations of natively unfolded peptides in the presence of explicit aqueous solvation, we observe that residue-specific P(II) conformational propensity is the result of the modulation of polypeptide backbone hydration by a proximal side-chain. Such a mechanism may be unique among those that contribute to the modulation of secondary structures in proteins. The calculated conformational propensities should prove useful for the development of a configurational P(II) scale necessary for the prediction and design of natural-like polypeptides.     

Conformational properties of a peptide model for unfolded alpha-helices

Models of protein folding often hypothesize that the first step is local secondary structure formation. The assumption is that unfolded polypeptide chains possess an intrinsic propensity to form these local secondary structures. On the basis of this idea, it is tempting to model the local conformational properties of unfolded proteins using well-established residue secondary structure propensities, in particular, alpha-helix forming propensities. We have used spectroscopic methods to investigate the conformational behavior of a host-guest series of peptides designed to model unfolded alpha-helices. A suitable peptide model for unfolded alpha-helices was determined from studies of the length dependence of the conformational properties of alanine-based peptides. The chosen host peptide possessed a small, detectable, alpha-helix content. Substituting various representative guest residues into the central position of the host peptide at times changed the conformational behavior dramatically, and often in ways that could not be predicted from known alpha-helix forming propensities. The data presented can be used to rationalize some of these propensities. However, it is clear that secondary structure propensities cannot be used to predict the local conformational properties of unfolded proteins.     

Properties of polyproline II, a secondary structure element implicated in protein-protein interactions

The polyproline II (PPII) conformation of protein backbone is an important secondary structure type. It is unusual in that, due to steric constraints, its main-chain hydrogen-bond donors and acceptors cannot easily be satisfied. It is unable to make local hydrogen bonds, in a manner similar to that of alpha-helices, and it cannot easily satisfy the hydrogen-bonding potential of neighboring residues in polyproline conformation in a manner analogous to beta-strands. Here we describe an analysis of polyproline conformations using the HOMSTRAD database of structurally aligned proteins. This allows us not only to determine amino acid propensities from a much larger database than previously but also to investigate conservation of amino acids in polyproline conformations, and the conservation of the conformation itself. Although proline is common in polyproline helices, helices without proline represent 46% of the total. No other amino acid appears to be greatly preferred; glycine and aromatic amino acids have low propensities for PPII. Accordingly, the hydrogen-bonding potential of PPII main-chain is mainly satisfied by water molecules and by other parts of the main-chain. Side-chain to main-chain interactions are mostly nonlocal. Interestingly, the increased number of nonsatisfied H-bond donors and acceptors (as compared with alpha-helices and beta-strands) makes PPII conformers well suited to take part in protein-protein interactions.     

A comprehensive library of blocked dipeptides reveals intrinsic backbone conformational propensities of unfolded proteins

Despite prolonged scientific efforts to elucidate the intrinsic peptide backbone preferences of amino-acids based on understanding of intermolecular forces, many open questions remain, particularly concerning neighboring peptide interaction effects on the backbone conformational distribution of short peptides and unfolded proteins. Here, we show that spectroscopic studies of a complete library of 400 dipeptides reveal that, irrespective of side-chain properties, the backbone conformation distribution is narrow and they adopt polyproline II and β-strand, indicating the importance of backbone peptide solvation and electronic effects. By directly comparing the dipeptide circular dichroism and NMR results with those of unfolded proteins, the comprehensive dipeptides form a complete set of structural motifs of unfolded proteins. We thus anticipate that the present dipeptide library with spectroscopic data can serve as a useful database for understanding the nature of unfolded protein structures and for further refinements of molecular mechanical parameters.     

Conformational analysis of short polar side-chain amino-acids through umbrella sampling and DFT calculations

Molecular and quantum mechanics calculations were carried out in a series of tripeptides (GXG, where X?=?D, N and C) as models of the unfolded states of proteins. The selected central amino acids, especially aspartic acid (D) and asparagine (N) are known to present significant average conformations in partially allowed areas of the Ramachandran plot, which have been suggested to be important in unfolded protein regions. In this report, we present the calculation of the propensity values through an umbrella sampling procedure in combination with the calculation of the NMR J-coupling constants obtained by a DFT model. The experimental NMR observations can be reasonably explained in terms of a conformational distribution where PP_II and β basins sum up propensities above 0.9. The conformational analysis of the side chain dihedral angle (χ₁), along with the computation of ³J(H^αH^β), revealed a preference for the g _? and g ₊ rotamers. These may be connected with the presence of intermolecular H-bonding and carbonyl–carbonyl interactions sampled in the PP_II and β basins. Taking into account all those results, it can be established that these residues show a similar behavior to other amino acids in short peptides regarding backbone φ,ψ dihedral angle distribution, in agreement with some experimental analysis of capped dipeptides.     

Polyproline II helix conformation in a proline-rich environment: a theoretical study

Interest centers here on whether a polyproline II helix can propagate through adjacent non-proline residues, and on shedding light on recent experimental observations suggesting the presence of significant PP(II) structure in a short alanine-based peptide with no proline in the sequence. For this purpose, we explored the formation of polyproline II helices in proline-rich peptides with the sequences Ac-(Pro)(3)-X-(Pro)(3)-Gly-Tyr-NH(2), with X = Pro (PPP), Ala (PAP), Gln (PQP), Gly (PGP), and Val (PVP), and Ac-(Pro)(3)-Ala-Ala-(Pro)(3)-Gly-Tyr-NH(2) (PAAP), by using a theoretical approach that includes a solvent effect as well as cis <--> trans isomerization of the peptide groups and puckering conformations of the pyrrolidine ring of the proline residues. Since (13)C chemical shifts have proven to be useful for identifying secondary-structure preferences in proteins and peptides, and because values of the dihedral angles (phi,psi) are the main determinants of their magnitudes, we have, therefore, computed the Boltzmann-averaged (13)C chemical shifts for the guest residues in the PXP peptide (X = Pro, Ala, Gln, Gly, and Val) with a combination of approaches, involving molecular mechanics, statistical mechanics, and quantum mechanics. In addition, an improved procedure was used to carry out the conformational searches and to compute the solvent polarization effects faster and more accurately than in previous work. The current theoretical work and additional experimental evidence show that, in short proline-rich peptides, alanine decreases the polyproline II helix content. In particular, the theoretical evidence accumulated in this work calls into question the proposal that alanine has a strong preference to adopt conformations in the polyproline II region of the Ramachandran map.     

Hydrodynamic Radii of Intrinsically Disordered Proteins Determined from Experimental Polyproline II Propensities

The properties of disordered proteins are thought to depend on intrinsic conformational propensities for polyproline II (PP _II) structure. While intrinsic PP _II propensities have been measured for the common biological amino acids in short peptides, the ability of these experimentally determined propensities to quantitatively reproduce structural behavior in intrinsically disordered proteins (IDPs) has not been established. Presented here are results from molecular simulations of disordered proteins showing that the hydrodynamic radius (R _h) can be predicted from experimental PP _II propensities with good agreement, even when charge-based considerations are omitted. The simulations demonstrate that R _h and chain propensity for PP _II structure are linked via a simple power-law scaling relationship, which was tested using the experimental R _h of 22 IDPs covering a wide range of peptide lengths, net charge, and sequence composition. Charge effects on R _h were found to be generally weak when compared to PP _II effects on R _h. Results from this study indicate that the hydrodynamic dimensions of IDPs are evidence of considerable sequence-dependent backbone propensities for PP _II structure that qualitatively, if not quantitatively, match conformational propensities measured in peptides.     

Sparsely populated residue conformations in protein structures: Revisiting “experimental” Ramachandran maps

The Ramachandran map clearly delineates the regions of accessible conformational (φ–ψ) space for amino acid residues in proteins. Experimental distributions of φ, ψ values in high‐resolution protein structures, reveal sparsely populated zones within fully allowed regions and distinct clusters in apparently disallowed regions. Conformational space has been divided into 14 distinct bins. Residues adopting these relatively rare conformations are presented and amino acid propensities for these regions are estimated. Inspection of specific examples in a completely “arid”, fully allowed region in the top left quadrant establishes that side‐chain and backbone interactions may provide the energetic compensation necessary for populating this region of φ–ψ space. Asn, Asp, and His residues showed the highest propensities in this region. The two distinct clusters in the bottom right quadrant which are formally disallowed on strict steric considerations correspond to the gamma turn (C7 axial) conformation (Bin 12 ) and the i + 1 position of Type II′ β turns (Bin 13) . Of the 516 non‐Gly residues in Bin 13 , 384 occupied the i + 1 position of Type II′ β turns. Further examination of these turn segments revealed a high propensity to occur at the N‐terminus of helices and as a tight turn in β hairpins. The β strand–helix motif with the Type II′ β turn as a connecting element was also found in as many as 57 examples. Proteins 2014; 82:1101–1112. © 2013 Wiley Periodicals, Inc.     

Folding type-specific secondary structure propensities of amino acids,derived from α-helical, β-sheet, α/β, and α+β proteins of known structures

Folding type-specific secondary structure propensities of 20 naturally occurring amino acids have been derived from α-helical, β-sheet, α/β, and α+β proteins of known structures. These data show that each residue type of amino acids has intrinsic propensities in different regions of secondary structures for different folding types of proteins. Each of the folding types shows markedly different rank ordering, indicating folding type-specific effects on the secondary structure propensities of amino acids. Rigorous statistical tests have been made to validate the folding type-specific effects. It should be noted that α and β proteins have relatively small α-helices and β-strands forming propensities respectively compared with those of α+β and α/β proteins. This may suggest that, with more complex architectures than α and β proteins, α+β and α/β proteins require larger propensities to distinguish from interacting α-helices and β-strands. Our finding of folding type-specific secondary structure propensities suggests that sequence space accessible to each folding type may have differing features. Differing sequence space features might be constrained by topological requirement for each of the folding types. Almost all strong β-sheet forming residues are hydrophobic in character regardless of folding types, thus suggesting the hydrophobicities of side chains as a key determinant of β-sheet structures. In contrast, conformational entropy of side chains is a major determinant of the helical propensities of amino acids, although other interactions such as hydrophobicities and charged interactions cannot be neglected. These results will be helpful to protein design, class-based secondary structure prediction, and protein folding. © 1998 John Wiley & Sons, Inc. Biopoly 45: 35–49, 1998     

Conformational distributions of denatured and unstructured proteins are similar to those of 20?×?20 blocked dipeptides

Understanding intrinsic conformational preferences of amino-acids in unfolded proteins is important for elucidating the underlying principles of their stability and re-folding on biological timescales. Here, to investigate the neighbor interaction effects on the conformational propensities of amino-acids, we carried out (1)H NMR experiments for a comprehensive set of blocked dipeptides and measured the scalar coupling constants between alpha protons and amide protons as well as their chemical shifts. Detailed inspection of these NMR properties shows that, irrespective of amino-acid side-chain properties, the distributions of the measured coupling constants and chemical shifts of the dipeptides are comparatively narrow, indicating small variances of their conformation distributions. They are further compared with those of blocked amino-acids (Ac-X-NHMe), oligopeptides (Ac-GGXGG-NH(2)), and native (lysozyme), denatured (lysozyme and outer membrane protein X from Escherichia coli), unstructured (Domain 2 of the protein 5A of Hepatitis C virus), and intrinsically disordered (hNlg3cyt: intracellular domain of human NL3) proteins. These comparative investigations suggest that the conformational preferences and local solvation environments of the blocked dipeptides are quite similar to not only those of other short oligopeptides but also those of denatured and natively unfolded proteins.     

Reconciling observations of sequence-specific conformational propensities with the generic polymeric behavior of denatured proteins

Radii of gyration of denatured proteins vary with chain length and are insensitive to details of amino acid sequence. Observations of sequence independence in polymeric properties conflict with results from spectroscopic experiments, which suggest the presence of sequence-specific residual structure in denatured states. Can we reconcile the two apparently conflicting sets of observations? To answer this question, we need knowledge of the ensemble of conformations accessible to proteins in good solvents. The excluded-volume limit provides an ideal mimic of polymers in good solvents. Therefore, we attempt to solve the "reconciliation problem" by simulating conformational ensembles accessible to peptides and proteins in the excluded-volume limit. Analysis of these ensembles for a variety of polypeptide sequences leads to results that are consistent with experimental observations of sequence-specific conformational preferences in short peptides and the scaling behavior of polymeric quantities for denatured proteins. Reconciliation in the excluded-volume limit comes about due to a tug of war between two factors, namely, minimization of steric overlap and the competing effects of conformational entropy. Minimization of steric overlap promotes chain stretching and leads to experimentally observed sequence-dependent preferences for locally extended segments such as polyproline II helices, beta-strands, and very short stretches of alpha-helix. Conformational entropy opposes chain stretching, and the calculated persistence length for sequence-dependent conformational preferences is less than five amino acids. This estimate does not vary with amino acid sequence. The short persistence lengths lead directly to experimental observations of generic sequence-independent behavior of radii of gyration for denatured proteins.     

Amino acid propensities for the collagen triple-helix

Determination of the tendencies of amino acids to form alpha-helical and beta-sheet structures has been important in clarifying stabilizing interactions, protein design, and the protein folding problem. In this study, we have determined for the first time a complete scale of amino acid propensities for another important protein motif: the collagen triple-helix conformation with its Gly-X-Y repeating sequence. Guest triplets of the form Gly-X-Hyp and Gly-Pro-Y are used to quantitate the conformational propensities of all 20 amino acids for the X and Y positions in the context of a (Gly-Pro-Hyp)(8) host peptide. The rankings for both the X and Y positions show the highly stabilizing nature of imino acids and the destabilizing effects of Gly and aromatic residues. Many residues show differing propensities in the X versus Y position, related to the nonequivalence of these positions in terms of interchain interactions and solvent exposure. The propensity of amino acids to adopt a polyproline II-like conformation plays a role in their triple-helix rankings, as shown by a moderate correlation of triple-helix propensity with frequency of occurrence in polyproline II-like regions. The high propensity of ionizable residues in the X position suggests the importance of interchain hydrogen bonding directly or through water to backbone carbonyls or hydroxyprolines. The low propensity of side chains with branching at the C(delta) in the Y position supports models suggesting these groups block solvent access to backbone C=O groups. These data provide a first step in defining sequence-dependent variations in local triple-helix stability and binding, and are important for a general understanding of side chain interactions in all proteins.