An amino acid code for irregular and mixed protein packing

To advance our understanding of protein tertiary structure, the development of the knob‐socket model is completed in an analysis of the packing in irregular coil and turn secondary structure packing as well as between mixed secondary structure. The knob‐socket model simplifies packing based on repeated patterns of two motifs: a three‐residue socket for packing within secondary (2°) structure and a four‐residue knob‐socket for tertiary (3°) packing. For coil and turn secondary structure, knob‐sockets allow identification of a correlation between amino acid composition and tertiary arrangements in space. Coil contributes almost as much as α‐helices to tertiary packing. In irregular sockets, Gly, Pro, Asp, and Ser are favored, while in irregular knobs, the preference order is Arg, Asp, Pro, Asn, Thr, Leu, and Gly. Cys, His,Met, and Trp are not favored in either. In mixed packing, the knob amino acid preferences are a function of the socket that they are packing into, whereas the amino acid composition of the sockets does not depend on the secondary structure of the knob. A unique motif of a coil knob with an XYZ β‐sheet socket may potentially function to inhibit β‐sheet extension. In addition, analysis of the preferred crossing angles for strands within a β‐sheet and mixed α‐helice/β‐sheet identifies canonical packing patterns useful in protein design. Lastly, the knob‐socket model abstracts the complexity of protein tertiary structure into an intuitive packing surface topology map. Proteins 2015; 83:2147–2161. © 2015 Wiley Periodicals, Inc.     

An amino acid code to define a protein's tertiary packing surface

One difficult aspect of the protein‐folding problem is characterizing the nonspecific interactions that define packing in protein tertiary structure. To better understand tertiary structure, this work extends the knob‐socket model by classifying the interactions of a single knob residue packed into a set of contiguous sockets, or a pocket made up of 4 or more residues. The knob‐socket construct allows for a symbolic two‐dimensional mapping of pockets. The two‐dimensional mapping of pockets provides a simple method to investigate the variety of pocket shapes to understand the geometry of protein tertiary surfaces. The diversity of pocket geometries can be organized into groups of pockets that share a common core, which suggests that some interactions in pockets are ancillary to packing. Further analysis of pocket geometries displays a preferred configuration that is right‐handed in α‐helices and left‐handed in β‐sheets. The amino acid composition of pockets illustrates the importance of nonpolar amino acids in packing as well as position specificity. As expected, all pocket shapes prefer to pack with hydrophobic knobs; however, knobs are not selective for the pockets they pack. Investigating side‐chain rotamer preferences for certain pocket shapes uncovers no strong correlations. These findings allow a simple vocabulary based on knobs and sockets to describe protein tertiary packing that supports improved analysis, design, and prediction of protein structure. Proteins 2016; 84:201–216. © 2015 Wiley Periodicals, Inc.     

Structural properties of amyloid β(1‐40) dimer explored by replica exchange molecular dynamics simulations

Replica exchange molecular dynamics simulations (300 ns) were used to study the dimerization of amyloid β(1‐40) (Aβ(1‐40)) polypeptide. Configurational entropy calculations revealed that at physiological temperature (310 K, 37°C) dynamic dimers are formed by randomly docked monomers. Free energy of binding of the two chains to each other was ?93.56 ± 6.341 kJ mol^?1. Prevalence of random coil conformations was found for both chains with the exceptions of increased β‐sheet content from residues 16‐21 and 29‐32 of chain A and residues 15‐21 and 30‐33 of chain B with β‐turn/β‐bend conformations in both chains from residues 1‐16, 21‐29 of chain A, 1‐16, and 21‐29 of chain B. There is a mixed β‐turn/β‐sheet region from residues 33‐38 of both chains. Analysis of intra‐ and interchain residue distances shows that, although the individual chains are highly flexible, the dimer system stays in a loosely packed antiparallel β‐sheet configuration with contacts between residues 17‐21 of chain A with residues 17‐21 and 31‐36 of chain B as well as residues 31‐36 of chain A with residues 17‐21 and 31‐36 of chain B. Based on dihedral principal component analysis, the antiparallel β‐sheet‐loop‐β‐sheet conformational motif is favored for many low energy sampled conformations. Our results show that Aβ(1‐40) can form dynamic dimers in aqueous solution that have significant conformational flexibility and are stabilized by collapse of the central and C‐terminal hydrophobic cores with the expected β‐sheet‐loop‐β‐sheet conformational motif. Proteins 2017; 85:1024–1045. © 2017 Wiley Periodicals, Inc.     

Crystal structure of a tripeptide containing aminocyclododecane carboxylic acid: a supramolecular twisted parallel β‐sheet in crystals

The crystal structure of a tripeptide Boc‐Leu‐Val‐Ac₁₂c‐OMe ( 1 ) is determined, which incorporates a bulky 1‐aminocyclododecane‐1‐carboxylic acid (Ac₁₂c) side chain. The peptide adopts a semi‐extended backbone conformation for Leu and Val residues, while the backbone torsion angles of the C^α,α‐dialkylated residue Ac₁₂c are in the helical region of the Ramachandran map. The molecular packing of 1 revealed a unique supramolecular twisted parallel β‐sheet coiling into a helical architecture in crystals, with the bulky hydrophobic Ac₁₂c side chains projecting outward the helical column. This arrangement resembles the packing of peptide helices in crystal structures. Although short oligopeptides often assemble as parallel or anti‐parallel β‐sheet in crystals, twisted or helical β‐sheet formation has been observed in a few examples of dipeptide crystal structures. Peptide 1 presents the first example of a tripeptide showing twisted β‐sheet assembly in crystals. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Sequence determinants of thermodynamic stability in a WW domain—An all‐β‐sheet protein

The stabilities of 66 sequence variants of the human Pin1 WW domain have been determined by equilibrium thermal denaturation experiments. All 34 residues composing the hPin1 WW three‐stranded β‐sheet structure could be replaced one at a time with at least one different natural or non‐natural amino acid residue without leading to an unfolded protein. Alanine substitutions at only four positions within the hPin1 WW domain lead to a partially or completely unfolded protein—in the absence of a physiological ligand. The side chains of these four residues form a conserved, partially solvent‐inaccessible, continuous hydrophobic minicore comprising the N‐ and C‐termini. Ala mutations at five other residues, three of which constitute the ligand binding patch on the concave side of the β‐sheet, significantly destabilize the hPin1 WW domain without leading to an unfolded protein. The remaining mutations affect protein stability only slightly, suggesting that only a small subset of side chain interactions within the hPin1 WW domain are mandatory for acquiring and maintaining a stable, cooperatively folded β‐sheet structure.     

A hierarchical order within protein structures underlies large separations between strands in β‐sheets

Protein β‐sheets often involve nonlocal interactions between parts of the polypeptide chain that are separated by hundreds of residues, raising the question of how these nonlocal contacts form. A recent study of the smallest β‐sheets found that their formation was not driven by signals hidden in the primary sequence. Instead, the strands in these sheets were either local in sequence, or, when separated by large sequential distances, the intervening residues were found to fold into compact modules that anchored distant parts of the chain in close spatial proximity. Here, we examine larger β‐sheets to investigate the extensibility of this principle. From an analysis of the β‐sheets in a nonredundant protein dataset, we find that a highly ordered hierarchical relationship exists in the intervening structure between nonlocal β‐strands. This observation is almost universal: virtually all β‐sheets, no matter their complexity, appear to adopt an antiparallel model to manage the nonlocal aspects of their assembly, one where the chain, having left the vicinity of an unfinished β‐sheet, retraces its steps via the same route to complete the initial sheet. Exceptions typically involve unstructured regions at chain termini. Moreover, an analysis of the residues involved in nonlocal crossstrand interactions did not produce any evidence of a signal hidden in the sequence that might direct long‐range interactions. These results build on those reported for the smallest sheets, suggesting that sheet formation is either local in sequence or local in space following prior folding events that anchor disparate parts of the chain in close proximity. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

An amino acid packing code for α-helical structure and protein design

This work demonstrates that all packing in α-helices can be simplified to repetitive patterns of a single motif: the knob-socket. Using the precision of Voronoi Polyhedra/Delauney Tessellations to identify contacts, the knob-socket is a four-residue tetrahedral motif: a knob residue on one α-helix packs into the three-residue socket on another α-helix. The principle of the knob-socket model relates the packing between levels of protein structure: the intra-helical packing arrangements within secondary structure that permit inter-helix tertiary packing interactions. Within an α-helix, the three-residue sockets arrange residues into a uniform packing lattice. Inter-helix packing results from a definable pattern of interdigitated knob-socket motifs between two α-helices. Furthermore, the knob-socket model classifies three types of sockets: (1) free, favoring only intra-helical packing; (2) filled, favoring inter-helical interactions; and (3) non, disfavoring α-helical structure. The amino acid propensities in these three socket classes essentially represent an amino acid code for structure in α-helical packing. Using this code, we used a novel yet straightforward approach for the design of α-helical structure to validate the knob-socket model. Unique sequences for three peptides were created to produce a predicted amount of α-helical structure: mostly helical, some helical, and no helix. These three peptides were synthesized, and helical content was assessed using CD spectroscopy. The measured α-helicity of each peptide was consistent with the expected predictions. These results and analysis demonstrate that the knob-socket motif functions as the basic unit of packing and presents an intuitive tool to decipher the rules governing packing in protein structure.     

Mechanism of formation of the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin binding protein G from Streptococcus. I. Importance of hydrophobic interactions in stabilization of β‐hairpin structure

We previously studied a 16‐amino acid‐residue fragment of the C‐terminal β‐hairpin of the B3 domain (residues 46–61), [IG(46–61)] of the immunoglobulin binding protein G from Streptoccocus, and found that hydrophobic interactions and the turn region play an important role in stabilizing the structure. Based on these results, we carried out systematic structural studies of peptides derived from the sequence of IG (46–61) by systematically shortening the peptide by one residue at a time from both the C‐ and the N‐terminus. To determine the structure and stability of two resulting 12‐ and 14‐amino acid‐residue peptides, IG(48–59) and IG(47–60), respectively, we carried out circular dichroism, NMR, and calorimetric studies of these peptides in pure water. Our results show that IG(48–59) possesses organized three‐dimensional structure stabilized by hydrophobic interactions (Tyr50–Phe57 and Trp48–Val59) at T = 283 and 305 K. At T = 313 K, the structure breaks down because of increased chain entropy, but the turn region is preserved in the same position observed for the structure of the whole protein. The breakdown of structure occurs near the melting temperature of this peptide (T_m = 310 K) measured by differential scanning calorimetry (DSC). The melting temperature of IG(47–60) determined by DSC is T_m = 330 K and its structure is similar to that of the native β‐hairpin at all (lower) temperatures examined (283–313 K). Both of these truncated sequences are conserved in all known amino acid sequences of the B domains of the immunoglobulin binding protein G from bacteria. Thus, this study contributes to an understanding of the mechanism of folding of this whole family of proteins, and provides information about the mechanism of formation and stabilization of a β‐hairpin structural element. Proteins 2009. © 2008 Wiley‐Liss, Inc.     

BuildBeta—A system for automatically constructing beta sheets

We describe a method that can thoroughly sample a protein conformational space given the protein primary sequence of amino acids and secondary structure predictions. Specifically, we target proteins with β‐sheets because they are particularly challenging for ab initio protein structure prediction because of the complexity of sampling long‐range strand pairings. Using some basic packing principles, inverse kinematics (IK), and β‐pairing scores, this method creates all possible β‐sheet arrangements including those that have the correct packing of β‐strands. It uses the IK algorithms of ProteinShop to move α‐helices and β‐strands as rigid bodies by rotating the dihedral angles in the coil regions. Our results show that our approach produces structures that are within 4–6 Å RMSD of the native one regardless of the protein size and β‐sheet topology although this number may increase if the protein has long loops or complex α‐helical regions. Proteins 2010. © Published 2009 Wiley‐Liss, Inc.     

Optimization of a β‐sheet‐cap for long loop closure

Protein loops make up a large portion of the secondary structure in nature. But very little is known concerning loop closure dynamics and the effects of loop composition on fold stability. We have designed a small system with stable β‐sheet structures, including features that allow us to probe these questions. Using paired Trp residues that form aromatic clusters on folding, we are able to stabilize two β‐strands connected by varying loop lengths and composition (an example sequence: R W ITVTI – loop – KKIRV W E). Using NMR and CD, both fold stability and folding dynamics can be investigated for these systems. With the 16 residue loop peptide (sequence: R W ITVTI‐(GGGGKK)₂GGGG‐KKIRV W E) remaining folded (ΔG_U = 1.6 kJ/mol at 295K). To increase stability and extend the series to longer loops, we added an additional Trp/Trp pair in the loop flanking position. With this addition to the strands, the 16 residue loop (sequence: R W ITVRI W ‐(GGGGKK)₂GGGG‐ W KTIRV W E) supports a remarkably stable β‐sheet (ΔG_U = 6.3 kJ/mol at 295 K, T_m = ～55°C). Given the abundance of loops in binding motifs and between secondary structures, these constructs can be powerful tools for peptide chemists to study loop effects; with the Trp/Trp pair providing spectroscopic probes for assessing both stability and dynamics by NMR.     

β‐Bulges: Extensive structural analyses of β‐sheets irregularities

β‐Sheets are quite frequent in protein structures and are stabilized by regular main‐chain hydrogen bond patterns. Irregularities in β‐sheets, named β‐bulges, are distorted regions between two consecutive hydrogen bonds. They disrupt the classical alternation of side chain direction and can alter the directionality of β‐strands. They are implicated in protein‐protein interactions and are introduced to avoid β‐strand aggregation. Five different types of β‐bulges are defined. Previous studies on β‐bulges were performed on a limited number of protein structures or one specific family. These studies evoked a potential conservation during evolution. In this work, we analyze the β‐bulge distribution and conservation in terms of local backbone conformations and amino acid composition. Our dataset consists of 66 times more β‐bulges than the last systematic study (Chan et al. Protein Science 1993, 2:1574–1590). Novel amino acid preferences are underlined and local structure conformations are highlighted by the use of a structural alphabet. We observed that β‐bulges are preferably localized at the N‐ and C‐termini of β‐strands, but contrary to the earlier studies, no significant conservation of β‐bulges was observed among structural homologues. Displacement of β‐bulges along the sequence was also investigated by Molecular Dynamics simulations.     

Helix‐sheet packing in proteins

The three‐dimensional structure of a protein is organized around the packing of its secondary structure elements. Although much is known about the packing geometry observed between α‐helices and between β‐sheets, there has been little progress on characterizing helix–sheet interactions. We present an analysis of the conformation of αβ₂ motifs in proteins, corresponding to all occurrences of helices in contact with two strands that are hydrogen bonded. The geometry of the αβ₂ motif is characterized by the azimuthal angle θ between the helix axis and an average vector representing the two strands, the elevation angle ψ between the helix axis and the plane containing the two strands, and the distance D between the helix and the strands. We observe that the helix tends to align to the two strands, with a preference for an antiparallel orientation if the two strands are parallel; this preference is diminished for other topologies of the β‐sheet. Side‐chain packing at the interface between the helix and the strands is mostly hydrophobic, with a preference for aliphatic amino acids in the strand and aromatic amino acids in the helix. From the knowledge of the geometry and amino acid propensities of αβ₂ motifs in proteins, we have derived different statistical potentials that are shown to be efficient in picking native‐like conformations among a set of non‐native conformations in well‐known decoy datasets. The information on the geometry of αβ₂ motifs as well as the related statistical potentials have applications in the field of protein structure prediction. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Boosting protein stability with the computational design of β‐sheet surfaces

β‐sheets often have one face packed against the core of the protein and the other facing solvent. Mutational studies have indicated that the solvent‐facing residues can contribute significantly to protein stability, and that the preferred amino acid at each sequence position is dependent on the precise structure of the protein backbone and the identity of the neighboring amino acids. This suggests that the most advantageous methods for designing β‐sheet surfaces will be approaches that take into account the multiple energetic factors at play including side chain rotamer preferences, van der Waals forces, electrostatics, and desolvation effects. Here, we show that the protein design software Rosetta, which models these energetic factors, can be used to dramatically increase protein stability by optimizing interactions on the surfaces of small β‐sheet proteins. Two design variants of the β‐sandwich protein from tenascin were made with 7 and 14 mutations respectively on its β‐sheet surfaces. These changes raised the thermal midpoint for unfolding from 45°C to 64°C and 74°C. Additionally, we tested an empirical approach based on increasing the number of potential salt bridges on the surfaces of the β‐sheets. This was not a robust strategy for increasing stability, as three of the four variants tested were unfolded.     

Alanine substitutions of noncysteine residues in the cysteine‐stabilized αβ motif

The protein scaffold is a peptide framework with a high tolerance of residue modifications. The cysteine‐stabilized αβ motif (CSαβ) consists of an α‐helix and an antiparallel triple‐stranded β‐sheet connected by two disulfide bridges. Proteins containing this motif share low sequence identity but high structural similarity and has been suggested as a good scaffold for protein engineering. The Vigna radiate defensin 1 (VrD1), a plant defensin, serves here as a model protein to probe the amino acid tolerance of CSαβ motif. A systematic alanine substitution is performed on the VrD1. The key residues governing the inhibitory function and structure stability are monitored. Thirty‐two of 46 residue positions of VrD1 are altered by site‐directed mutagenesis techniques. The circular dichroism spectrum, intrinsic fluorescence spectrum, and chemical denaturation are used to analyze the conformation and structural stability of proteins. The secondary structures were highly tolerant to the amino acid substitutions; however, the protein stabilities were varied for each mutant. Many mutants, although they maintained their conformations, altered their inhibitory function significantly. In this study, we reported the first alanine scan on the plant defensin containing the CSαβ motif. The information is valuable to the scaffold with the CSαβ motif and protein engineering.     

PackHelix: a tool for helix-sheet packing during protein structure prediction

The three‐dimensional structure of a protein is organized around the packing of its secondary structure elements. Predicting the topology and constructing the geometry of structural motifs involving α‐helices and/or β‐strands are therefore key steps for accurate prediction of protein structure. While many efforts have focused on how to pack helices and on how to sample exhaustively the topologies and geometries of multiple strands forming a β‐sheet in a protein, there has been little progress on generating native‐like packings of helices on sheets. We describe a method that can generate the packing of multiple helices on a given β‐sheet for αβα sandwich type protein folds. This method mines the results of a statistical analysis of the conformations of αβ₂ motifs in protein structures to provide input values for the geometric attributes of the packing of a helix on a sheet. It then proceeds with a geometric builder that generates multiple arrangements of the helices on the sheet of interest by sampling through these values and performing consistency checks that guarantee proper loop geometry between the helices and the strands, minimal number of collisions between the helices, and proper formation of a hydrophobic core. The method is implemented as a module of ProteinShop. Our results show that it produces structures that are within 4–6 Å RMSD of the native one, regardless of the number of helices that need to be packed, though this number may increase if the protein has several helices between two consecutive strands in the sequence that pack on the sheet formed by these two strands. Proteins 2011; Published 2011 Wiley‐Liss, Inc.     

Dissecting the roles of local packing density and longer‐range effects in protein sequence evolution

What are the structural determinants of protein sequence evolution? A number of site‐specific structural characteristics have been proposed, most of which are broadly related to either the density of contacts or the solvent accessibility of individual residues. Most importantly, there has been disagreement in the literature over the relative importance of solvent accessibility and local packing density for explaining site‐specific sequence variability in proteins. We show that this discussion has been confounded by the definition of local packing density. The most commonly used measures of local packing, such as contact number and the weighted contact number, represent the combined effects of local packing density and longer‐range effects. As an alternative, we propose a truly local measure of packing density around a single residue, based on the Voronoi cell volume. We show that the Voronoi cell volume, when calculated relative to the geometric center of amino‐acid side chains, behaves nearly identically to the relative solvent accessibility, and each individually can explain, on average, approximately 34% of the site‐specific variation in evolutionary rate in a data set of 209 enzymes. An additional 10% of variation can be explained by nonlocal effects that are captured in the weighted contact number. Consequently, evolutionary variation at a site is determined by the combined effects of the immediate amino‐acid neighbors of that site and effects mediated by more distant amino acids. We conclude that instead of contrasting solvent accessibility and local packing density, future research should emphasize on the relative importance of immediate contacts and longer‐range effects on evolutionary variation. Proteins 2016; 84:841–854. © 2016 Wiley Periodicals, Inc.     

Determination of the secondary structural elements of chicken liver fatty acid binding protein by two‐dimensional homonuclear NMR

A conformational study in solution of the fatty acid binding protein from chicken liver is presented. The nearly complete sequence‐specific ¹H resonance assignment was achieved from homonuclear two‐dimensional nmr experiments using a sample of native protein. The principal elements of secondary structure were identified: 10 antiparallel β‐strands and one helical segment followed by a turn comprising 5 residues. These elements correspond closely with those of the crystal structure of the related protein, and two new secondary structural features obtained from the nmr data are the β‐sheet conformation between the first and the last β‐strand in the protein sequence, as well as a helical loop at the N‐terminus of the polypeptide chain. © 1999 John Wiley & Sons, Inc. Biopoly 50: 1–11, 1999     

The effect of β‐methylation on the conformation of α, β‐dehydrophenylalanine: a DFT study

Dehydroamino acids are non‐coded amino acids that offer unique conformational properties. Dehydrophenylalanine (ΔPhe) is most commonly used to modify bioactive peptides to constrain the topography of the phenyl ring in the side chain, which commonly serves as a pharmacophore. The Ramachandran maps (in the gas phase and in CHCl₃ mimicking environments) of ΔPhe analogues with methyl groups at the β position of the side chain as well as at the C‐terminal amide were calculated using the B3LYP/6‐31 + G** method. Unexpectedly, β‐methylation alone results in an increase of conformational freedom of the affected ΔPhe residue. However, further modification by introducing an additional methyl group at C‐terminal methyl amide results in a steric crowding that fixes the torsion angle ψ of all conformers to the value 123°, regardless of the Z or E position of the phenyl ring. The number of conformers is reduced and the accessible conformational space of the residues is very limited. In particular, (Z)‐Δ(βMe)Phe with the tertiary C‐terminal amide can be classified as the amino acid derivative that has a single conformational state as it seems to adopt only the β conformation. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Observed octameric assembly of a Plasmodium yoelii peroxiredoxin can be explained by the replacement of native “ball‐and‐socket” interacting residues by an affinity tag

Peroxiredoxins (Prxs) are ubiquitous and efficient antioxidant enzymes crucial for redox homeostasis in most organisms, and are of special importance for disease‐causing parasites that must protect themselves against the oxidative weapons of the human immune system. Here, we describe reanalyses of crystal structures of two Prxs from malaria parasites. In addition to producing improved structures, we provide normalizing explanations for features that had been noted as unusual in the original report of these structures (Qiu et al., BMC Struct Biol 2012;12:2). Most importantly, we provide evidence that the unusual octameric assembly seen for Plasmodium yoelii Prx1a is not physiologically relevant, but arises because the structure is not of authentic P. yoelii Prx1a, but a variant we designate PyPrx1a^N* that has seven native N‐terminal residues replaced by an affinity tag. This N‐terminal modification disrupts a previously unrecognized, hydrophobic “ball‐and‐socket” interaction conserved at the B‐type dimer interface of Prx1 subfamily enzymes, and is accommodated by a fascinating two‐residue “β‐slip” type register shift in the β‐strand association at a dimer interface. The resulting change in the geometry of the dimer provides a simple explanation for octamer formation. This study illustrates how substantive impacts can occur in protein variants in which native residues have been altered.     

Mechanism of formation of the C‐terminal β‐hairpin of the B3 domain of the immunoglobulin‐binding protein G from Streptococcus. IV. Implication for the mechanism of folding of the parent protein

A 34‐residue α/β peptide [IG(28–61)], derived from the C‐terminal part of the B3 domain of the immunoglobulin binding protein G from Streptoccocus, was studied using CD and NMR spectroscopy at various temperatures and by differential scanning calorimetry. It was found that the C‐terminal part (a 16‐residue‐long fragment) of this peptide, which corresponds to the sequence of the β‐hairpin in the native structure, forms structure similar to the β‐hairpin only at T = 313 K, and the structure is stabilized by non‐native long‐range hydrophobic interactions (Val47–Val59). On the other hand, the N‐terminal part of IG(28–61), which corresponds to the middle α‐helix in the native structure, is unstructured at low temperature (283 K) and forms an α‐helix‐like structure at 305 K, and only one helical turn is observed at 313 K. At all temperatures at which NMR experiments were performed (283, 305, and 313 K), we do not observe any long‐range connectivities which would have supported packing between the C‐terminal (β‐hairpin) and the N‐terminal (α‐helix) parts of the sequence. Such interactions are absent, in contrast to the folding pathway of the B domain of protein G, proposed recently by Kmiecik and Kolinski (Biophys J 2008, 94, 726–736), based on Monte‐Carlo dynamics studies. Alternative folding mechanisms are proposed and discussed. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 469–480, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com