Analysis and design of turns in alpha-helical hairpins

Although the analysis and design of turns that connect the strands in antiparallel beta-hairpins has reached an advanced state, much less is known concerning turns between antiparallel helices in helical hairpins. We have conducted an analysis of the structures and sequence preferences of two types of interhelical turns, each of which connects the two helices by a two-residue linker in an alphaL-beta conformation. Based on this analysis, it became apparent that the turn introduced into a designed four-helix bundle protein, DF1, did not occur within an optimal structural context. DF1 is a dimeric model for the diiron class of proteins. A longer loop with a beta-alphaR-beta conformation was inserted between two helices in the protein, and a sequence was chosen to stabilize its conformation. X-ray crystallography and NMR analysis of the protein showed the structure to be in excellent agreement with design.     

Redesigning the type II' β‐turn in green fluorescent protein to type I': Implications for folding kinetics and stability

Both Type I' and Type II' β‐turns have the same sense of the β‐turn twist that is compatible with the β‐sheet twist. They occur predominantly in two residue β‐hairpins, but the occurrence of Type I' β‐turns is two times higher than Type II' β‐turns. This suggests that Type I' β‐turns may be more stable than Type II' β‐turns, and Type I' β‐turn sequence and structure can be more favorable for protein folding than Type II' β‐turns. Here, we redesigned the native Type II' β‐turn in GFP to Type I' β‐turn, and investigated its effect on protein folding and stability. The Type I' β‐turns were designed based on the statistical analysis of residues in natural Type I' β‐turns. The substitution of the native “GD” sequence of i+1 and i+2 residues with Type I' preferred “(N/D)G” sequence motif increased the folding rate by 50% and slightly improved the thermodynamic stability. Despite the enhancement of in vitro refolding kinetics and stability of the redesigned mutants, they showed poor soluble expression level compared to wild type. To overcome this problem, i and i + 3 residues of the designed Type I' β‐turn were further engineered. The mutation of Thr to Lys at i + 3 could restore the in vivo soluble expression of the Type I' mutant. This study indicates that Type II' β‐turns in natural β‐hairpins can be further optimized by converting the sequence to Type I'. Proteins 2014; 82:2812–2822. © 2014 Wiley Periodicals, Inc.     

Tight Turns of Outer Membrane Proteins: An Analysis of Sequence,Structure, and Hydrogen Bonding

As a structural class, tight turns can control molecular recognition, enzymatic activity, and nucleation of folding. They have been extensively characterized in soluble proteins but have not been characterized in outer membrane proteins (OMPs), where they also support critical functions. We clustered the 4 to 6 residue tight turns of 110 OMPs to characterize the phi/psi angles, sequence, and hydrogen bonding of these structures. We find significant differences between reports of soluble protein tight turns and OMP tight turns. Since OMP strands are less twisted than soluble strands, they favor different turn structures types. Moreover, the membrane localization of OMPs yields different sequence hallmarks for their tight turns relative to soluble protein turns. We also characterize the differences in phi/psi angles, sequence, and hydrogen bonding between OMP extracellular loops and OMP periplasmic turns. As previously noted, the extracellular loops tend to be much longer than the periplasmic turns. We find that this difference in length is due to the broader distribution of lengths of the extracellular loops not a large difference in the median length. Extracellular loops also tend to have more charged residues as predicted by the charge-out rule. Finally, in all OMP tight turns, hydrogen bonding between the side chain and backbone 2 to 4 residues away from that side chain plays an important role. These bonds preferentially use an Asp, Asn, Ser, or Thr residue in a beta or pro phi/psi conformation. We anticipate that this study will be applicable to future design and structure prediction of OMPs.     

Propensities of peptides containing the Asn‐Gly segment to form β‐turn and β‐hairpin structures

The propensities of peptides that contain the Asn‐Gly segment to form β‐turn and β‐hairpin structures were explored using the density functional methods and the implicit solvation model in CH₂Cl₂ and water. The populations of preferred β‐turn structures varied depending on the sequence and solvent polarity. In solution, β‐hairpin structures with βI′ turn motifs were most preferred for the heptapeptides containing the Asn‐Gly segment regardless of the sequence of the strands. These preferences in solution are consistent with the corresponding X‐ray structures. The sequence, H‐bond strengths, solvent polarity, and conformational flexibility appeared to interact to determine the preferred β‐hairpin structure of each heptapeptide, although the β‐turn segments played a role in promoting the formation of β‐hairpin structures and the β‐hairpin propensity varied. In the heptapeptides containing the Asn‐Gly segment, the β‐hairpin formation was enthalpically favored and entropically disfavored at 25°C in water. The calculated results for β‐turns and β‐hairpins containing the Asn‐Gly segment imply that these structural preferences may be useful for the design of bioactive macrocyclic peptides containing β‐hairpin mimics and the design of binding epitopes for protein–protein and protein–nucleic acid recognitions. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 653–664, 2016.     

Water and side‐chain embedded π‐turns

Elucidating protein function from its structure is central to the understanding of cellular mechanisms. This involves deciphering the dependence of local structural motifs on sequence. These structural motifs may be stabilized by direct or water‐mediated hydrogen bonding among the constituent residues. π‐Turns, defined by interactions between (i) and (i + 5) positions, are large enough to contain a central space that can embed a water molecule (or a protein moiety) to form a stable structure. This work is an analysis of such embedded π‐turns using a nonredundant dataset of protein structures. A total of 2965 embedded π‐turns have been identified, as also 281 embedded Schellman motif, a type of π‐turn which occurs at the C‐termini of α‐helices. Embedded π‐turns and Schellman motifs have been classified on the basis of the protein atoms of the terminal turn residues that are linked by the embedded moiety, conformation, residue composition, and compared with the turns that have terminal residues connected by direct hydrogen bonds. Geometrically, the turns have been fitted to a circle and the position of the linker relative to its center analyzed. The hydroxyl group of Ser and Thr, located at (i + 3) position, is the most prominent linker for the side‐chain mediated π‐turns. Consideration of residue conservation among homologous sequences indicates the terminal and the linker positions to be the most conserved. The embedded π‐turn as a binding site (for the linker) is discussed in the context of “nest,” a concave depression that is formed in protein structures with adjacent residues having enantiomeric main‐chain conformations. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 441–453, 2014.     

Turn scanning by site-directed mutagenesis: application to the protein folding problem using the intestinal fatty acid binding protein

We have systematically mutated residues located in turns between beta-strands of the intestinal fatty acid binding protein (IFABP), and a glycine in a half turn, to valine and have examined the stability, refolding rate constants and ligand dissociation constants for each mutant protein. IFABP is an almost all beta-sheet protein exhibiting a topology comprised of two five-stranded sheets surrounding a large cavity into which the fatty acid ligand binds. A glycine residue is located in seven of the eight turns between the antiparallel beta-strands and another in a half turn of a strand connecting the front and back sheets. Mutations in any of the three turns connecting the last four C-terminal strands slow the folding and decrease stability with the mutation between the last two strands slowing folding dramatically. These data suggest that interactions between the last four C-terminal strands are highly cooperative, perhaps triggered by an initial hydrophobic collapse. We suggest that this trigger is collapse of the highly hydrophobic cluster of amino acids in the D and E strands, a region previously shown to also affect the last stage of the folding process (Kim et al., 1997). Changing the glycine in the strand between the front and back sheets also results in a unstable, slow folding protein perhaps disrupting the D-E strand interactions. For most of the other turn mutations there was no apparent correlation between stability and refolding rate constants. In some turns, the interaction between strands, rather than the turn type, appears to be critical for folding while in others, turn formation itself appears to be a rate limiting step. Although there is no simple correlation between turn formation and folding kinetics, we propose that turn scanning by mutagenesis will be a useful tool for issues related to protein folding.     

Native structural propensity in cellular retinoic acid-binding protein I 64-88: the role of locally encoded structure in the folding of a beta-barrel protein

A central question in protein folding is the relative importance of locally encoded structure and cooperative interactions among residues distant in sequence. We have been exploring this question in a predominantly β-sheet protein, since β-structure formation clearly relies on both local and global sequence information. We present evidence that a 24-residue peptide corresponding to two linked hairpins of cellular retinoic acid-binding protein I (CRABP I) adopts significant native structure in aqueous solution. Prior work from our laboratory showed that the two turns contained in this fragment (turns III and IV) had the highest tendency of any of the eight turns in this anti-parallel β-barrel to fold into native turns. In addition, the primary sequence of these two turns is well conserved throughout the structural family to which CRABP I belongs, and residues in the turns and their associated hairpins participate in a network of conserved long-range interactions. We propose that the strong local-sequence biases within the chain segment comprising turns III and IV favor longer-range interactions that are crucial to the folding and native-state stability of CRABP I, and may play a similar role in related intracellular lipid-binding proteins (iLBPs).     

Folding of a beta-hairpin peptide derived from the N-terminus of ubiquitin. Conformational preferences of beta-turn residues dictate non-native beta-strand interactions.

The role of the non-native beta-turn sequence (NPDG) in nucleating the folding of a beta-hairpin peptide derived from the N-terminus of ubiquitin, has been examined by NMR and CD spectroscopy. The NPDG sequence, while representing a common two-residue type I turn sequence in proteins, folds to give a G1-bulged type I turn in the context of a beta-hairpin peptide, to the exclusion of other possible conformations. The turn conformation results in misalignment of the two beta strands and a beta hairpin with non-native side chain interactions. A truncated 12-residue analogue of the hairpin, in which the majority of residues in the N-terminal beta strand have been deleted, shows some weak propensity to fold into a G-bulged type I turn conformation in the absence of interstrand stabilizing interactions. The NPDG turn sequence pays some of the entropic cost in initiating folding allowing interstrand interactions, which in this case arise from the non-native pairing of residue side chains, to stabilize a significant population of the folded state. Examination of the relative abundance of the Pro-Asp type I turn, with G in the +B1 position, vs. the type I G-bulged turn PXG, in a database of high resolution structures, reveals 48 instances of PXG bulged turns for which X = Asp is by far the most common residue with 20 occurrences. Strikingly, there are no examples of a type I PD turn with G at the +B1 position, in good agreement with our experimental observations that the PDG G-bulged turn is populated preferentially in solution.     

β‐Hairpin folding and stability: molecular dynamics simulations of designed peptides in aqueous solution

The structural properties of a 10‐residue and a 15‐residue peptide in aqueous solution were investigated by molecular dynamics simulation. The two designed peptides, SYINSDGTWT and SESYINSDGTWTVTE, had been studied previously by NMR at 278 K and the resulting model structures were classified as 3:5 β‐hairpins with a type I + G1 β‐bulge turn. In simulations at 278 K, starting from the NMR model structure, the 3:5 β‐hairpin conformers proved to be stable over the time period evaluated (30 ns). Starting from an extended conformation, simulations of the decapeptide at 278 K, 323 K and 353 K were also performed to study folding. Over the relatively short time scales explored (30 ns at 278 K and 323 K, 56 ns at 353 K), folding to the 3:5 β‐hairpin could only be observed at 353 K. At this temperature, the collapse to β‐hairpin‐like conformations is very fast. The conformational space accessible to the peptide is entirely dominated by loop structures with different degrees of β‐hairpin character. The transitions between different types of ordered loops and β‐hairpins occur through two unstructured loop conformations stabilized by a single side‐chain interaction between Tyr2 and Trp9, which facilitates the changes of the hydrogen‐bond register. In agreement with previous experimental results, β‐hairpin formation is initially driven by the bending propensity of the turn segment. Nevertheless, the fine organization of the turn region appears to be a late event in the folding process. Copyright © 2004 European Peptide Society and John Wiley & Sons, Ltd.     

Structural motifs in which β‐strands are clipped together with the П‐like module

In this study, the structural motifs that can be represented as combinations of small motifs such as β‐hairpins, S‐, and Z‐like β‐sheets and βαβ‐units, and the П‐like module are described and analyzed. The П‐module consists of connected elements of the β‐strand‐loop‐β‐strand type arranged in space so that its overall fold resembles a clip or the Greek letter П. In proteins, the П‐module itself and the structural motifs containing it exhibit unique overall folds and have specific sequence patterns of the key hydrophobic, hydrophilic and glycine residues. All this together enables us to conclude that these structural motifs can fold independently of the remaining part of the molecule and can act as nuclei and/or “ready‐made” building blocks in protein folding.     

Recurrent alpha beta loop structures in TIM barrel motifs show a distinct pattern of conserved structural features.

A systematic survey of seven parallel alpha/beta barrel protein domains, based on exhaustive structural comparisons, reveals that a sizable proportion of the alpha beta loops in these proteins--20 out of a total of 49--belong to either one of two loop types previously described by Thornton and co-workers. Six loops are of the alpha beta 1 type, with one residue between the alpha-helix and beta-strand, and 13 are of the alpha beta 3 type, with three residues between the helix and the strand. Protein fragments embedding the identified loops, and termed alpha beta connections since they contain parts of the flanking helix and strand, have been analyzed in detail revealing that each type of connection has a distinct set of conserved structural features. The orientation of the beta-strand relative to the helix and loop portions is different owing to a very localized difference in backbone conformation. In alpha beta 1 connections, the chain enters the beta-strand via a residue adopting an extended conformation, while in alpha beta 3 it does so via a residue in a near alpha-helical conformation. Other conserved structural features include distinct patterns of side chain orientation relative to the beta-sheet surface and of main chain H-bonds in the loop and the beta-strand moieties. Significant differences also occur in packing interactions of conserved hydrophobic residues situated in the last turn of the helix. Yet the alpha-helix surface of both types of connections adopts similar orientations relative to the barrel sheet surface. Our results suggest furthermore that conserved hydrophobic residues along the sequence of the connections, may be correlated more with specific patterns of interactions made with neighboring helices and sheet strands than with helix/strand packing within the connection itself. A number of intriguing observations are also made on the distribution of the identified alpha beta 1 and alpha beta 3 loops within the alpha/beta-barrel motifs. They often occur adjacent to each other; alpha beta 3 loops invariably involve even numbered beta-strands, while alpha beta 1 loops involve preferentially odd beta-strands; all the analyzed proteins contain at least one alpha beta 3 loop in the first half of the eightfold alpha/beta barrel. Possible origins of all these observations, and their relevance to the stability and folding of parallel alpha/beta barrel motifs are discussed.     

Sequence swapping does not result in conformation swapping for the beta4/beta5 and beta8/beta9 beta-hairpin turns in human acidic fibroblast growth factor

The beta-turn is the most common type of nonrepetitive structure in globular proteins, comprising ~25% of all residues; however, a detailed understanding of effects of specific residues upon beta-turn stability and conformation is lacking. Human acidic fibroblast growth factor (FGF-1) is a member of the beta-trefoil superfold and contains a total of five beta-hairpin structures (antiparallel beta-sheets connected by a reverse turn). beta-Turns related by the characteristic threefold structural symmetry of this superfold exhibit different primary structures, and in some cases, different secondary structures. As such, they represent a useful system with which to study the role that turn sequences play in determining structure, stability, and folding of the protein. Two turns related by the threefold structural symmetry, the beta4/beta5 and beta8/beta9 turns, were subjected to both sequence-swapping and poly-glycine substitution mutations, and the effects upon stability, folding, and structure were investigated. In the wild-type protein these turns are of identical length, but exhibit different conformations. These conformations were observed to be retained during sequence-swapping and glycine substitution mutagenesis. The results indicate that the beta-turn structure at these positions is not determined by the turn sequence. Structural analysis suggests that residues flanking the turn are a primary structural determinant of the conformation within the turn.     

Very short peptides with stable folds: building on the interrelationship of Trp/Trp, Trp/cation, and Trp/backbone-amide interaction geometries

By combining a favorable turn sequence with a turn flanking Trp/Trp interaction and a C-terminal H-bonding interaction between a backbone amide and an i-2 Trp ring, a particularly stable (DeltaG(U) > 7 kJ/mol) truncated hairpin, Ac-WI-(D-Pro-D-Asn)-KWTG-NH(2), results. In this construct and others with a W-(4-residue turn)-W motif in severely truncated hairpins, the C-terminal Trp is the edge residue in a well-defined face-to-edge (FtE) aryl/aryl interaction. Longer hairpins and those with six-residue turns retain the reversed "edge-to-face" (EtF) Trp/Trp geometry first observed for the trpzip peptides. Mutational studies suggest that the W-(4-residue turn)-W interaction provides at least 3 kJ/mol of stabilization in excess of that due to the greater beta-propensity of Trp. The pi-cation, and Trp/Gly-H(N) interactions have been defined. The latter can give rise to >3 ppm upfield shifts for the Gly-H(N) in -WX(n)G- units both in turns (n = 2) and at the C-termini (n = 1) of hairpins. Terminal YTG units result in somewhat smaller shifts (extrapolated to 2 ppm for 100% folding). In peptides with both the EtF and FtE W/W interaction geometries, Trp to Tyr mutations indicate that Trp is the preferred "face" residue in aryl/aryl pairings, presumably because of its greater pi basicity.     

Measuring cooperativity in the formation of a three-stranded beta sheet (double hairpin)

Recently validated chemical shift measures of hairpin structuring have been applied to a series of turn mutants of the Schenck-Gellman three-strand beta-sheet model with the aim of measuring the entropic advantage associated with aligning an additional strand onto an existing hairpin versus aligning the same two strands in an initial hairpin formation. In a four-state analysis (unfolded, 2 single hairpins, and the double hairpin fold in equilibrium) a cooperativity index can be defined as the factor by which the equilibrium constant for hairpin formation is improved when one strand is prestructured. This cooperativity index is 2.7 +/- 0.7 for hairpin formation about a stable D-Pro-Gly turn locus and increases to 7.6 +/- 1.2 for an Asn-Gly turn locus. The latter corresponds to a cooperativity induced DeltaDeltaG increment of 4.9 kJ/mol for the folding of a hairpin. Although larger than previous experimental measures of folding cooperativity in three-stranded sheets, the magnitude of this effect (which is considerably less than the TDeltaDeltaS expectation for prestructuring three or more beta-strand residue sites) likely reflects the intrinsic preference of these designed sequences for extended conformations. If similar or larger effects apply to protein beta-sheet folding, it is not surprising that particularly favorable hairpin alignments serve as nucleation sites in protein folding pathways.     

ω‐Turn: A novel β‐turn mimic in globular proteins stabilized by main‐chain to side‐chain CH···O interaction

Mimicry of structural motifs is a common feature in proteins. The 10‐membered hydrogen‐bonded ring involving the main‐chain C?O in a β‐turn can be formed using a side‐chain carbonyl group leading to Asx‐turn. We show that the N? H component of hydrogen bond can be replaced by a C^γ‐H group in the side chain, culminating in a nonconventional C? H···O interaction. Because of its shape this β‐turn mimic is designated as ω‐turn, which is found to occur ～three times per 100 residues. Three residues (i to i + 2) constitute the turn with the C? H···O interaction occurring between the terminal residues, constraining the torsion angles ?_{i + 1}, ψ_{i + 1}, ?_{i + 2} and χ′_{1(i + 2)} (using the interacting C^γ atom). Based on these angles there are two types of ω‐turns, each of which can be further divided into two groups. C^β‐branched side‐chains, and Met and Gln have high propensities to occur at i + 2; for the last two residues the carbonyl oxygen may participate in an additional interaction involving the S and amino group, respectively. With Cys occupying the i + 1 position, such turns are found in the metal‐binding sites. N‐linked glycosylation occurs at the consensus pattern Asn‐Xaa‐Ser/Thr; with Thr at i + 2, the sequence can adopt the secondary structure of a ω‐turn, which may be the recognition site for protein modification. Location between two β‐strands is the most common occurrence in protein tertiary structure, and being generally exposed ω‐turn may constitute the antigenic determinant site. It is a stable scaffold and may be used in protein engineering and peptide design. Proteins 2015; 83:203–214. © 2014 Wiley Periodicals, Inc.     

Structural predictions of AgfA, the insoluble fimbrial subunit of Salmonella thin aggregative fimbriae.

The unusually stable and multifunctional, thin aggregative fimbriae common to all Salmonella spp. are principally polymers of the fimbrin subunit, AgfA. AgfA of Salmonella enteritidis consists of two domains: a protease-sensitive, 22 amino acid residue N-terminal region and a protease-resistant, 109 residue C-terminal core. The unusual amino acid sequence of the AgfA core region comprises two-, five- and tenfold internal sequence homology patterns reflected in five conserved, 18-residue tandem repeats. These repeats have the consensus sequence, Sx5QxGx2NxAx3Q and are linked together by four or five residues, (x)xAx2. The predicted secondary structure for this unusual arrangement of tandem repeats in AgfA indicates mainly extended conformation with the beta strands linked by four to six residues. Candidate proteins of known structure with motifs of alternating beta strands and short loops were selected from folds described in SCOP as a source of coordinates for AgfA model construction. Three all-beta class motifs selected from the Serratia marcescens metalloprotease, myelin P2 protein or vitelline membrane outer protein I were used for initial AgfA homology build-up procedures ultimately resulting in three structural models; beta barrel, beta prism and parallel beta helix. The beta barrel model is a compact, albeit irregular structure, with the beta strands arranged in two antiparallel beta sheet faces. The beta prism model does not reflect the 5 or 10-fold symmetry of the AgfA primary sequence. However, the favored, parallel beta helix model is a compact coil of ten helically arranged beta strands forming two parallel beta sheet faces. This arrangement predicts a regular, potentially stable, C-terminal core region consistent with the observed tandem repeat sequences, protease-resistance and strong tendency of this fimbrin to oligomerize and aggregate. Positional conservation of amino acid residues in AgfA and the Escherichia coli AgfA homologue, CsgA, provides strong support for this model. The parallel beta helix model of AgfA offers an interesting solution to a multifunctional fimbrin molecular surface having solvent exposed areas, regions for major and minor subunit interactions as well as fiber-fiber interactions common to many bacterial fimbriae.     

HMMSTR: a hidden Markov model for local sequence-structure correlations in proteins

We describe a hidden Markov model, HMMSTR, for general protein sequence based on the I-sites library of sequence-structure motifs. Unlike the linear hidden Markov models used to model individual protein families, HMMSTR has a highly branched topology and captures recurrent local features of protein sequences and structures that transcend protein family boundaries. The model extends the I-sites library by describing the adjacencies of different sequence-structure motifs as observed in the protein database and, by representing overlapping motifs in a much more compact form, achieves a great reduction in parameters. The HMM attributes a considerably higher probability to coding sequence than does an equivalent dipeptide model, predicts secondary structure with an accuracy of 74.3 %, backbone torsion angles better than any previously reported method and the structural context of beta strands and turns with an accuracy that should be useful for tertiary structure prediction.     

Physical�Cchemical determinants of coil conformations in globular proteins

We present a method with the potential to generate a library of coil segments from first principles. Proteins are built from α‐helices and/or β‐strands interconnected by these coil segments. Here, we investigate the conformational determinants of short coil segments, with particular emphasis on chain turns. Toward this goal, we extracted a comprehensive set of two‐, three‐, and four‐residue turns from X‐ray–elucidated proteins and classified them by conformation. A remarkably small number of unique conformers account for most of this experimentally determined set, whereas remaining members span a large number of rare conformers, many occurring only once in the entire protein database. Factors determining conformation were identified via Metropolis Monte Carlo simulations devised to test the effectiveness of various energy terms. Simulated structures were validated by comparison to experimental counterparts. After filtering rare conformers, we found that 98% of the remaining experimentally determined turn population could be reproduced by applying a hydrogen bond energy term to an exhaustively generated ensemble of clash‐free conformers in which no backbone polar group lacks a hydrogen‐bond partner. Further, at least 90% of longer coil segments, ranging from 5‐ to 20 residues, were found to be structural composites of these shorter primitives. These results are pertinent to protein structure prediction, where approaches can be divided into either empirical or ab initio methods. Empirical methods use database‐derived information; ab initio methods rely on physical–chemical principles exclusively. Replacing the database‐derived coil library with one generated from first principles would transform any empirically based method into its corresponding ab initio homologue.     

The ensemble folding kinetics of the FBP28 WW domain revealed by an all-atom Monte Carlo simulation in a knowledge-based potential

In this work, we apply a detailed all‐atom model with a transferable knowledge‐based potential to study the folding kinetics of Formin‐Binding protein, FBP28, which is a canonical three‐stranded β‐sheet WW domain. Replica exchange Monte Carlo simulations starting from random coils find native‐like (Cα RMSD of 2.68 Å) lowest energy structure. We also study the folding kinetics of FBP28 WW domain by performing a large number of ab initio Monte Carlo folding simulations. Using these trajectories, we examine the order of formation of two β‐hairpins, the folding mechanism of each individual β‐hairpin, and transition state ensemble (TSE) of FBP28 WW domain and compare our results with experimental data and previous computational studies. To obtain detailed structural information on the folding dynamics viewed as an ensemble process, we perform a clustering analysis procedure based on graph theory. Further, a rigorous P_fold analysis is used to obtain representative samples of the TSEs showing good quantitative agreement between experimental and simulated Φ values. Our analysis shows that the turn structure between first and second β strands is a partially stable structural motif that gets formed before entering the TSE in FBP28 WW domain and there exist two major pathways for the folding of FBP28 WW domain, which differ in the order and mechanism of hairpin formation. Proteins 2011. © 2011 Wiley‐Liss, Inc.     

Role of local sequence in the folding of cellular retinoic abinding protein I: structural propensities of reverse turns

The experiments described here explore the role of local sequence in the folding of cellular retinoic acid binding protein I (CRABP I). This is a 136-residue, 10-stranded, antiparallel beta-barrel protein with seven beta-hairpins and is a member of the intracellular lipid binding protein (iLBP) family. The relative roles of local and global sequence information in governing the folding of this class of proteins are not well-understood. In question is whether the beta-turns are locally defined by short-range interactions within their sequences, and are thus able to play an active role in reducing the conformational space available to the folding chain, or whether the turns are passive, relying upon global forces to form. Short (six- and seven-residue) peptides corresponding to the seven CRABP I turns were analyzed by circular dichroism and NMR for their tendencies to take up the conformations they adopt in the context of the native protein. The results indicate that two of the peptides, encompassing turns III and IV in CRABP I, have a strong intrinsic bias to form native turns. Intriguingly, these turns are on linked hairpins in CRABP I and represent the best-conserved turns in the iLBP family. These results suggest that local sequence may play an important role in narrowing the conformational ensemble of CRABP I during folding.