The sequence TGAAKAVALVL from glyceraldehyde-3-phosphate dehydrogenase displays structural ambivalence and interconverts between alpha-helical and beta-hairpin conformations mediated by collapsed conformational states.

The peptide TGAAKAVALVL from glyceraldehyde-3-phosphate dehydrogenase adopts a helical conformation in the crystal structure and is a site for two hydrated helical segments, which are thought to be helical folding intermediates. Overlapping sequences of four to five residues from the peptide, sample both helical and strand conformations in known protein structures, which are dissimilar to glyceraldehyde-3-phosphate dehydrogenase suggesting that the peptide may have a structural ambivalence. Molecular dynamics simulations of the peptide sequence performed for a total simulation time of 1.2 micros, starting from the various initial conformations using GROMOS96 force field under NVT conditions, show that the peptide samples a large number of conformational forms with transitions from alpha-helix to beta-hairpin and vice versa. The peptide, therefore, displays a structural ambivalence. The mechanism from alpha-helix to beta-hairpin transition and vice versa reveals that the compact bends and turns conformational forms mediate such conformational transitions. These compact structures including helices and hairpins have similar hydrophobic radius of gyration (Rgh) values suggesting that similar hydrophobic interactions govern these conformational forms. The distribution of conformational energies is Gaussian with helix sampling lowest energy followed by the hairpins and coil. The lowest potential energy of the full helix may enable the peptide to take up helical conformation in the crystal structure of the glyceraldehyde-3-phosphate dehydrogenase, even though the peptide has a preference for hairpin too. The relevance of folding and unfolding events observed in our simulations to hydrophobic collapse model of protein folding are discussed.     

Location of helical regions in tetrapyrrole-containing proteins by a helical hydrophobic moment analysis. Application to phytochrome

Helical regions in many tetrapyrrole proteins are highly amphiphilic, one side interacting with a hydrophobic core and another side interacting with the polar solvent. The mean helical hydrophobic moment is a measure of amphiphilicity of a helix. Helical regions in myoglobin, the alpha and beta subunits of C-phycocyanin, and cytochrome c can be distinguished from nonhelical regions by use of a hydrophobic moment analysis. 24 of 27 (89%) of the helical regions in these proteins were located by this analysis. Calculations were also performed on chymotrypsin, ribonuclease, and papain, which do not possess as pronounced a hydrophobic core as the tetrapyrrole-containing proteins. Less than 50% of the helical regions were correctly located, indicating a lack of amphiphilicity in the helices of these proteins. The hydrophobic moment analysis was also used to predict helical regions in phytochrome, the ubiquitous photoreceptor in plants. Additionally, this analysis is used to quickly locate internal hydrophilic residues which may be functionally important. The distribution of hydrophobic moments from a random sequence was determined so that qualitative and to some extent quantitative comparisons between different amphiphilic helices may be made.     

Helix packing moments reveal diversity and conservation in membrane protein structure

Helical membrane proteins are more tightly packed and the packing interactions are more diverse than those found in helical soluble proteins. Based on a linear correlation between amino acid packing values and interhelical propensity, we propose the concept of a helix packing moment to predict the orientation of helices in helical membrane proteins and membrane protein complexes. We show that the helix packing moment correlates with the helix interfaces of helix dimers of single pass membrane proteins of known structure. Helix packing moments are also shown to help identify the packing interfaces in membrane proteins with multiple transmembrane helices, where a single helix can have multiple contact surfaces. Analyses are described on class A G protein-coupled receptors (GPCRs) with seven transmembrane helices. We show that the helix packing moments are conserved across the class A family of GPCRs and correspond to key structural contacts in rhodopsin. These contacts are distinct from the highly conserved signature motifs of GPCRs and have not previously been recognized. The specific amino acid types involved in these contacts, however, are not necessarily conserved between subfamilies of GPCRs, indicating that the same protein architecture can be supported by a diverse set of interactions. In GPCRs, as well as membrane channels and transporters, amino acid residues with small side-chains (Gly, Ala, Ser, Cys) allow tight helix packing by mediating strong van der Waals interactions between helices. Closely packed helices, in turn, facilitate interhelical hydrogen bonding of both weakly polar (Ser, Thr, Cys) and strongly polar (Asn, Gln, Glu, Asp, His, Arg, Lys) amino acid residues. We propose the use of the helix packing moment as a complementary tool to the helical hydrophobic moment in the analysis of transmembrane sequences.     

Folding of Aquaporin 1: Multiple evidence that helix 3 can shift out of the membrane core

The folding of most integral membrane proteins follows a two‐step process: initially, individual transmembrane helices are inserted into the membrane by the Sec translocon. Thereafter, these helices fold to shape the final conformation of the protein. However, for some proteins, including Aquaporin 1 (AQP1), the folding appears to follow a more complicated path. AQP1 has been reported to first insert as a four‐helical intermediate, where helix 2 and 4 are not inserted into the membrane. In a second step, this intermediate is folded into a six‐helical topology. During this process, the orientation of the third helix is inverted. Here, we propose a mechanism for how this reorientation could be initiated: first, helix 3 slides out from the membrane core resulting in that the preceding loop enters the membrane. The final conformation could then be formed as helix 2, 3, and 4 are inserted into the membrane and the reentrant regions come together. We find support for the first step in this process by showing that the loop preceding helix 3 can insert into the membrane. Further, hydrophobicity curves, experimentally measured insertion efficiencies and MD‐simulations suggest that the barrier between these two hydrophobic regions is relatively low, supporting the idea that helix 3 can slide out of the membrane core, initiating the rearrangement process.     

Importance of context in protein folding: secondary structural propensities versus tertiary contact-assisted secondary structure formation

Molecular dynamics simulations can be used to reveal the detailed conformational behaviors of peptides and proteins. By comparing fragment and full-length protein simulations, we can investigate the role of each peptide segment in the folding process. Here, we take advantage of information regarding the helix formation process from our previous simulations of barnase and protein A as well as new simulations of four helical fragments from these proteins at three different temperatures, starting with both helical and extended structures. Segments with high helical propensity began the folding process by tethering the chain through side chain interactions involving either polar interactions, such as salt bridges, or hydrophobic staples. These tethers were frequently nonnative (i.e., not i --> i + 4 spacing) and provided a scaffold for other residues, thereby limiting the conformational search. The helical structure then propagated on both sides of the tether. Segments with low stability and propensity formed later in the folding process and utilized contacts with other portions of the protein when folding. These helices formed via a tertiary contact-assisted mechanism, primarily via hydrophobic contacts between residues distant in sequence. Thus, segments with different helical propensities appear to play different roles during protein folding. Furthermore, the active role of nonlocal side chains in helix formation highlights why we must move beyond simple hierarchical models of protein folding.     

Comparison of helix interactions in membrane and soluble alpha-bundle proteins

Helix-helix interactions are important for the folding, stability, and function of membrane proteins. Here, two independent and complementary methods are used to investigate the nature and distribution of amino acids that mediate helix-helix interactions in membrane and soluble alpha-bundle proteins. The first method characterizes the packing density of individual amino acids in helical proteins based on the van der Waals surface area occluded by surrounding atoms. We have recently used this method to show that transmembrane helices pack more tightly, on average, than helices in soluble proteins. These studies are extended here to characterize the packing of interfacial and noninterfacial amino acids and the packing of amino acids in the interfaces of helices that have either right- or left-handed crossing angles, and either parallel or antiparallel orientations. We show that the most abundant tightly packed interfacial residues in membrane proteins are Gly, Ala, and Ser, and that helices with left-handed crossing angles are more tightly packed on average than helices with right-handed crossing angles. The second method used to characterize helix-helix interactions involves the use of helix contact plots. We find that helices in membrane proteins exhibit a broader distribution of interhelical contacts than helices in soluble proteins. Both helical membrane and soluble proteins make use of a general motif for helix interactions that relies mainly on four residues (Leu, Ala, Ile, Val) to mediate helix interactions in a fashion characteristic of left-handed helical coiled coils. However, a second motif for mediating helix interactions is revealed by the high occurrence and high average packing values of small and polar residues (Ala, Gly, Ser, Thr) in the helix interfaces of membrane proteins. Finally, we show that there is a strong linear correlation between the occurrence of residues in helix-helix interfaces and their packing values, and discuss these results with respect to membrane protein structure prediction and membrane protein stability.     

Structural comparison and classification of alpha‐helical transmembrane domains based on helix interaction patterns

Structural classification of membrane proteins is still in its infancy due to the relative paucity of available three‐dimensional structures compared with soluble proteins. However, recent technological advances in protein structure determination have led to a significant increase in experimentally known membrane protein folds, warranting exploration of the structural universe of membrane proteins. Here, a new and completely membrane protein specific structural classification system is introduced that classifies α‐helical membrane proteins according to common helix architectures. Each membrane protein is represented by a helix interaction graph depicting transmembrane helices with their pairwise interactions resulting from individual residue contacts. Subsequently, proteins are clustered according to similarities among these helix interaction graphs using a newly developed structural similarity score called HISS. As HISS scores explicitly disregard structural properties of loop regions, they are more suitable to capture conserved transmembrane helix bundle architectures than other structural similarity scores. Importantly, we are able to show that a classification approach based on helix interaction similarity closely resembles conventional structural classification databases such as SCOP and CATH implying that helix interactions are one of the major determinants of α‐helical membrane protein folds. Furthermore, the classification of all currently available membrane protein structures into 20 recurrent helix architectures and 15 singleton proteins demonstrates not only an impressive variability of membrane helix bundles but also the conservation of common helix interaction patterns among proteins with distinctly different sequences. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Exploring the propensities of helices in PrP(C) to form beta sheet using NMR structures and sequence alignments

Neurodegenerative diseases induced by transmissible spongiform encephalopathies are associated with prions. The most spectacular event in the formation of the infectious scrapie form, referred to as PrP(Sc), is the conformational change from the predominantly alpha-helical conformation of PrP(C) to the PrP(Sc) state that is rich in beta-sheet content. Using sequence alignments and structural analysis of the available nuclear magnetic resonance structures of PrP(C), we explore the propensities of helices in PrP(C) to be in a beta-strand conformation. Comparison of a number of structural characteristics (such as solvent accessible area, distribution of (Phi, Psi) angles, mismatches in hydrogen bonds, nature of residues in local and nonlocal contacts, distribution of regular densities of amino acids, clustering of hydrophobic and hydrophilic residues in helices) between PrP(C) structures and a databank of "normal" proteins shows that the most unusual features are found in helix 2 (H2) (residues 172-194) followed by helix 1 (H1) (residues 144-153). In particular, the C-terminal residues in H2 are frustrated in their helical state. The databank of normal proteins consists of 58 helical proteins, 36 alpha+beta proteins, and 31 beta-sheet proteins. Our conclusions are also substantiated by gapless threading calculations that show that the normalized Z-scores of prion proteins are similar to those of other alpha+beta proteins with low helical content. Application of the recently introduced notion of discordance, namely, incompatibility of the predicted and observed secondary structures, also points to the frustration of H2 not only in the wild type but also in mutants of human PrP(C). This suggests that the instability of PrP(C) proteins may play a role in their being susceptible to the profound conformational change. Our analysis shows that, in addition to the previously proposed role for the segment (90-120) and possibly H1, the C-terminus of H2 and possibly N-terminus may play a role in the alpha-->beta transition. An implication of our results is that the ease of polymerization depends on the unfolding rate of the monomer. Sequence alignments show that helices in avian prion proteins (chicken, duck, crane) are better accommodated in a helical state, which might explain the absence of PrP(Sc) formation over finite time scales in these species. From this analysis, we predict that correlated mutations that reduce the frustration in the second half of helix 2 in mammalian prion proteins could inhibit the formation of PrP(Sc).     

Peptide design: Crystal structure of a helical peptide module attached to a potentially nonhelical amino terminal segment

The peptide Boc-Gly-Dpg-Gly-Val-Ala-Leu-Aib-Val-Ala-Leu-OMe has been designed to examine the structural consequences of placing a short segment with a low helix propensity at the amino terminus of a helical heptapeptide module. The Gly-Dpg-Gly segment is a potential connecting element in the synthetic construction of a helix-linker-helix motif. Crystal parameters for the peptide are P2₁, a = 8.651(3) Å, b = 46.826(13) Å, c = 16.245 Å, β = 90.13(3)*, Z = 4; 2 independent molecules/asymmetric unit. The structure reveals almost identical conformations for the two independent molecules. The backbone is completely helical for residues 2–9, with one 4 → 1 hydrogen bond and six 5 → 1 hydrogen bonds. The α,α-di-n-propylglycine residue adopts a helical conformation. Gly(1) adopts an extended conformation resulting in a nonhelical N-terminus, with the Boc group swinging away from the helix. The lateral association of helices in the b axis direction is unusual in that the helix axes are directed up or down (parallel or antiparallel) by pairs: ↓↓↑↑↓↓, etc. © 1996 John Wiley & Sons, Inc.     

Iterative assembly of helical proteins by optimal hydrophobic packing

We present a method for the computer-based iterative assembly of native-like tertiary structures of helical proteins from alpha-helical fragments. For any pair of helices, our method, called MATCHSTIX, first generates an ensemble of possible relative orientations of the helices with various ways to form hydrophobic contacts between them. Those conformations having steric clashes, or a large radius of gyration of hydrophobic residues, or with helices too far separated to be connected by the intervening linking region, are discarded. Then, we attempt to connect the two helical fragments by using a robotics-based loop-closure algorithm. When loop closure is feasible, the algorithm generates an ensemble of viable interconnecting loops. After energy minimization and clustering, we use a representative set of conformations for further assembly with the remaining helices, adding one helix at a time. To efficiently sample the conformational space, the order of assembly generally proceeds from the pair of helices connected by the shortest loop, followed by joining one of its adjacent helices, always proceeding with the shorter connecting loop. We tested MATCHSTIX on 28 helical proteins each containing up to 5 helices and found it to heavily sample native-like conformations. The average rmsd of the best conformations for the 17 helix-bundle proteins that have 2 or 3 helices is less than 2 A; errors increase somewhat for proteins containing more helices. Native-like states are even more densely sampled when disulfide bonds are known and imposed as restraints. We conclude that, at least for helical proteins, if the secondary structures are known, this rapid rigid-body maximization of hydrophobic interactions can lead to small ensembles of highly native-like structures. It may be useful for protein structure prediction.     

Conformational studies on concentrated solutions of poly(gamma-benzyl L-glutamate)

We have studied the effect of polypeptide concentration on the helix–coil transition of poly(γ-benzyl L -glutamate) (PBLG) in both dichloroacetic acid (DCA) and DCA–chloroform (CHF) mixtures. In agreement with other reports, we find the van't Hoff transition enthalpy to be strongly dependent on PBLG concentration. Also, an apparent effect of polypeptide concentration was noted on the transition temperature; however, corrections for finite PBLG concentration on the mole fraction of DCA seem to remove this effect. In order to explain our data, as well as some calorimetric data in the literature, we consider the transition free energy and enthalpy as a sum of three partial terms. These represent the thermodynamic parameters associated with: (1) conformational changes of the polypeptide, e.g. formation or disruption of intramolecular hydrogen bonds; (2) binding by the strong acid to the nonhelical segments of the polypeptide; (3) an overall (weak) interaction of the polypeptide with the nonbound solvent giving rise to dilution parameters that are dependent on the polypeptide conformation. The latter effect is generally ignored, since it is assumed that solvent interactions, other than specific binding, are similar for both the helical and the nonhelical conformation. Striking effects of water (small amounts) and solution aging on the formation of PBLG helices was observed. Water, as expected, acts as a helicogenic solvent when combined with DCA. The processes occurring during solution aging are not known, although the net effect is to stabilize the helical conformation. Finally, we present some rather unique thermally induced transitions of concentrated PBLG (about 200 mg/ml) in DCA. At low temperatures the soluble randomly coiled conformation is present. Heating produces first an isotropic gel, followed at higher temperatures by an isotropic solution consisting of about 70% α-helicity.     

Thermodynamic parameters of the helix-coil transition in polypeptide chains. III. Random copolymers of L-leucine and L-glutamic acid.

Conformational transitions induced by pH changes in random copolymers of leucine and glutamic acid have been studied. Significant differences were observed in the potentiometric titration curves of copolymers with small (up to 4%) and large leucine contents. The helical stability of copolymers with small leucine content, determined from titration curves by the Zimm and Rice method, decreases slightly with an increase in the leucine content, whereas the helical stability of copolymers with large leucine content increases sharply with an increase of the leucine content. It is shown that copolymers with large leucine content aggregate in the region of transition into the helical state, but the increase of their helical state stability is not connected with intermolecular aggregation, as it was also observed for a nonaggregating fraction isolated from one of the copolymers by gel chromatography. A conclusion is made that the helix–coil equilibrium constant s for leucine does not itself exceed the s constant for uncharged polyglutamic acid. The stabilization of the helical state in copolymers with large leucine content is due to intramolecular aggregation of helices in these copolymers. The analysis of the leucine residue distribution between helical and nonhelical regions in globular proteins also gives no real arguments to ascribe special helix-forming properties to leucine.     

Diffusing and colliding: the atomic level folding/unfolding pathway of a small helical protein

Proteins with ultra-fast folding/unfolding kinetics are excellent candidates for study by molecular dynamics. Here, we describe such simulations of a three helix bundle protein, the engrailed homeodomain (En-HD), which folds via the diffusion-collision model. The unfolding pathway of En-HD was characterized by seven simulations of the protein and 12 simulations of its helical fragments yielding over 1.1 micros of simulation time in water. Various conformational states along the unfolding pathway were identified. There is the compact native-like transition state, a U-shaped helical intermediate and an unfolded state with dynamic helical segments. Each of these states is in good agreement with experimental data. Examining these states as well as the transitions between them, we find the role of long-range tertiary contacts, specifically salt-bridges, important in the folding/unfolding pathway. In the folding direction, charged residues form long-range tertiary contacts before the hydrophobic core is formed. The formation of HII is assisted by a specific salt-bridge and by non-specific (fluctuating) tertiary contacts, which we call contact-assisted helix formation. Salt-bridges persist as the protein approaches the transition state, stabilizing HII until the hydrophobic core is formed. To complement this information, simulations of fragments of En-HD illustrate the helical propensities of the individual segments. By thermal denaturation, HII proved to be the least stable helix, unfolding in less than 450 ps at high temperature. We observed the low helical propensity of C-terminal residues from HIII in fragment simulations which, when compared to En-HD unfolding simulations, link the unraveling of HIII to the initial event that drives the unfolding of En-HD.     

Sequence and conformational preferences at termini of α‐helices in membrane proteins: Role of the helix environment

α‐helices are amongst the most common secondary structural elements seen in membrane proteins and are packed in the form of helix bundles. These α‐helices encounter varying external environments (hydrophobic, hydrophilic) that may influence the sequence preferences at their N and C‐termini. The role of the external environment in stabilization of the helix termini in membrane proteins is still unknown. Here we analyze α‐helices in a high‐resolution dataset of integral α‐helical membrane proteins and establish that their sequence and conformational preferences differ from those in globular proteins. We specifically examine these preferences at the N and C‐termini in helices initiating/terminating inside the membrane core as well as in linkers connecting these transmembrane helices. We find that the sequence preferences and structural motifs at capping (Ncap and Ccap) and near‐helical (N' and C') positions are influenced by a combination of features including the membrane environment and the innate helix initiation and termination property of residues forming structural motifs. We also find that a large number of helix termini which do not form any particular capping motif are stabilized by formation of hydrogen bonds and hydrophobic interactions contributed from the neighboring helices in the membrane protein. We further validate the sequence preferences obtained from our analysis with data from an ultradeep sequencing study that identifies evolutionarily conserved amino acids in the rat neurotensin receptor. The results from our analysis provide insights for the secondary structure prediction, modeling and design of membrane proteins. Proteins 2014; 82:3420–3436. © 2014 Wiley Periodicals, Inc.     

The role of helix stabilizing residues in GCN4 basic region folding and DNA binding

Basic region leucine zipper (bZip) proteins contain a bipartite DNA-binding motif consisting of a coiled-coil leucine zipper dimerization domain and a highly charged basic region that directly contacts DNA. The basic region is largely unfolded in the absence of DNA, but adopts a helical conformation upon DNA binding. Although a coil --> helix transition is entropically unfavorable, this conformational change positions the DNA-binding residues appropriately for sequence-specific interactions with DNA. The N-terminal residues of the GCN4 DNA-binding domain, DPAAL, make no DNA contacts and are not part of the conserved basic region, but are nonetheless important for DNA binding. Asp and Pro are often found at the N-termini of alpha-helices, and such N-capping motifs can stabilize alpha-helical structure. In the present study, we investigate whether these two residues serve to stabilize a helical conformation in the GCN4 basic region, lowering the energetic cost for DNA binding. Our results suggest that the presence of these residues contributes significantly to helical structure and to the DNA-binding ability of the basic region in the absence of the leucine zipper. Similar helix-capping motifs are found in approximately half of all bZip domains, and the implications of these findings for in vivo protein function are discussed.     

Conformational analysis by NMR and distance geometry techniques of a peptide mimetic of the third helix of the Antennapedia homeodomain.

The Antennapedia homeodomain structure consists of four helices. The helices II and III are connected by a tripeptide that forms a turn, and constitute the well-known helix-turn-helix motif. The recognition helix penetrates the DNA major groove, gives specific protein-DNA contacts and forms direct, or water-mediated, intermolecular hydrogen bonds. It was suggested that helix III (and perhaps also helix IV) might represent the recognition helix of Antennapedia homeodomain, which makes contact with the surface of the major groove of the DNA. In an attempt to clarify the helix III capabilities of assuming an helical conformation when separated from the rest of the protein, we carried out the structural determination of the recognition helix III in different solvent media. The conformational study of fragments 42-53, where residues W48 and F49, not involved in the protein-DNA interaction, were substituted by two alanines, was conducted in sodium dodecyl sulfate (SDS), trifluoroethanol (TFE) and TFE/water, using circular dichroism, nuclear magnetic resonance (NMR) and distance geometry (DG) techniques. The fragment assumes a well-defined secondary structure in TFE and in TFE/water (90/10, v/v) with an alpha-helix encompassing residues 4-9, while in TFE/water (70/30, v/v) a less regular structure was found. The DG results in the micellar system evidence the presence of a distorted alpha-helical conformation involving residues 4-8. Our results reveal that the isolated Antennapedia recognition helix III tend to preserve in solution the alpha-helical conformation even if separated from the rest of the molecule.     

Conformational features of a hexapeptide model Ac-TGAAKA-NH2 corresponding to a hydrated alpha helical segment from glyceraldehyde 3-phosphate dehydrogenase: implications for the role of turns in helix folding

Recent analysis of alpha helices in protein crystal structures, available in literature, revealed hydrated alpha helical segments in which, water molecule breaks open helix 5-->1 hydrogen bond by inserting itself, hydrogen bonds to both C=O and NH groups of helix hydrogen bond without disrupting the helix hydrogen bond, and hydrogen bonds to either C=O or NH of helix hydrogen bond. These hydrated segments display a variety of turn conformations and are thought to be 'folding intermediates' trapped during folding-unfolding of alpha helices. A role for reverse turns is implicated in the folding of alpha helices. We considered a hexapeptide model Ac-1TGAAKA6-NH2 from glyceraldehyde 3-phosphate dehydrogenase, corresponding to a hydrated helical segment to assess its role in helix folding. The sequence is a site for two 'folding intermediates'. The conformational features of the model peptide have been investigated by 1H 2D NMR techniques and quantum mechanical perturbative configuration interaction over localized orbitals (PCILO) method. Theoretical modeling largely correlates with experimental observations. Based upon the amide proton temperature coefficients, the observed d alpha n(i, i + 1), d alpha n(i, i + 2), dnn(i, i + 1), d beta n(i, i + 1) NOEs and the results from theoretical modeling, we conclude that the residues of the peptide sample alpha helical and neck regions of the Ramachandran phi, psi map with reduced conformational entropy and there is a potential for turn conformations at N and C terminal ends of the peptide. The role of reduced conformational entropy and turn potential in helix formation have been discussed. We conclude that the peptide sequence can serve as a 'folding intermediate' in the helix folding of glyceraldehyde 3-phosphate dehydrogenase.     

Characteristic features of amino acid residues in coiled-coil protein structures

Detailed analyses of protein structures provide an opportunity to understand conformation and function in terms of amino acid sequence and composition. In this work, we have systematically analyzed the characteristic features of the amino acid residues found in alpha-helical coiled-coils and, in so doing, have developed indices for their properties, conformational parameters, surrounding hydrophobicity and flexibility. As expected, there is preference for hydrophobic (Ala, Leu), positive (Lys, Arg) and negatively (Glu) charged residues in coiled-coil domains. However, the surrounding hydrophobicity of residues in coiled-coil domains is significantly less than that for residues in other regions of coiled-coil proteins. The analysis of temperature factors in coiled-coil proteins shows that the residues in these domains are more stable than those in other regions. Further, we have delineated the medium- and long-range contacts in coiled-coil domains and compared the results with those obtained for other (non-coiled-coil) parts of the same proteins and non-coiled-coil helical segments of globular proteins. The residues in coiled-coil domains are largely influenced by medium-range contacts, whereas long-range interactions play a dominant role in other regions of these same proteins as well as in non-coiled-coil helices. We have also revealed the preference of amino acid residues to form cation-pi interactions and we found that Arg is more likely to form such interactions than Lys. The parameters developed in this work can be used to understand the folding and stability of coiled-coil proteins in general.     

Design of stable α‐helices using global sequence optimization

The rational design of peptide and protein helices is not only of practical importance for protein engineering but also is a useful approach in attempts to improve our understanding of protein folding. Recent modifications of theoretical models of helix‐coil transitions allow accurate predictions of the helix stability of monomeric peptides in water and provide new possibilities for protein design. We report here a new method for the design of α‐helices in peptides and proteins using AGADIR, the statistical mechanical theory for helix‐coil transitions in monomeric peptides and the tunneling algorithm of global optimization of multidimensional functions for optimization of amino acid sequences. CD measurements of helical content of peptides with optimized sequences indicate that the helical potential of protein amino acids is high enough to allow formation of stable α‐helices in peptides as short as of 10 residues in length. The results show the maximum achievable helix content (HC) of short peptides with fully optimized sequences at 5 °C is expected to be ～70–75%. Under certain conditions the method can be a powerful practical tool for protein engineering. Unlike traditional approaches that are often used to increase protein stability by adding a few favorable interactions to the protein structure, this method deals with all possible sequences of protein helices and selects the best one from them. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.