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.     

Factors involved in the stability of isolated beta-sheets: Turn sequence, beta-sheet twisting, and hydrophobic surface burial

We have recently reported on the design of a 20-residue peptide able to form a significant population of a three-stranded up-and-down antiparallel beta-sheet in aqueous solution. To improve our beta-sheet model in terms of the folded population, we have modified the sequences of the two 2-residue turns by introducing the segment DPro-Gly, a sequence shown to lead to more rigid type II' beta-turns. The analysis of several NMR parameters, NOE data, as well as Deltadelta(CalphaH), DeltadeltaC(beta), and Deltadelta(Cbeta) values, demonstrates that the new peptide forms a beta-sheet structure in aqueous solution more stable than the original one, whereas the substitution of the DPro residues by LPro leads to a random coil peptide. This agrees with previous results on beta-hairpin-forming peptides showing the essential role of the turn sequence for beta-hairpin folding. The well-defined beta-sheet motif calculated for the new designed peptide (pair-wise RMSD for backbone atoms is 0.5 +/- 0.1 A) displays a high degree of twist. This twist likely contributes to stability, as a more hydrophobic surface is buried in the twisted beta-sheet than in a flatter one. The twist observed in the up-and-down antiparallel beta-sheet motifs of most proteins is less pronounced than in our designed peptide, except for the WW domains. The additional hydrophobic surface burial provided by beta-sheet twisting relative to a "flat" beta-sheet is probably more important for structure stability in peptides and small proteins like the WW domains than in larger proteins for which there exists a significant contribution to stability arising from their extensive hydrophobic cores.     

Effects of turn residues in directing the formation of the beta-sheet and in the stability of the beta-sheet

The designed peptide (denoted 20-mer, sequence VFITS(D)PGKTYTEV(D)PGOKILQ) has been shown to form a three-strand antiparallel beta-sheet. It is generally believed that the (D)Pro-Gly segment has the propensity to adopt a type II' beta-turn, thereby promoting the formation of this beta-sheet. Here, we replaced (D)Pro-Gly with Asp-Gly, which should favor a type I' turn, to examine the influence of different type of turns on the stability of the beta-sheet. Contrary to our expectation, the mutant peptide, denoted P6D, forms a five-residue type I turn plus a beta-bulge between the first two strands due to a one amino-acid frameshift in the hydrogen bonding network and side-chain inversion of the first beta-strand. In contrast, the same kind of substitution at (D)Pro-14 in the double mutant, denoted P6DP14D, does not yield the same effect. These observations suggest that the SDGK sequence disfavors the type I' conformation while the VDGO sequence favors a type I' turn, and that the frameshift in the first strand provides a way for the peptide to accommodate a disfavored turn sequence by protruding a bulge in the formation of the beta-hairpin. Thus, different types of turns can affect the stability of a beta-structure.     

Beta-hairpin formation in aqueous solution and in the presence of trifluoroethanol: a (1)H and (13)C nuclear magnetic resonance conformational study of designed peptides

In order to check our current knowledge on the principles involved in beta-hairpin formation, we have modified the sequence of a 3:5 beta-hairpin forming peptide with two different purposes, first to increase the stability of the formed 3:5 beta-hairpin, and second to convert the 3:5 beta-hairpin into a 2:2 beta-hairpin. The conformational behavior of the designed peptides was investigated in aqueous solution and in 30% trifluoroethanol (TFE) by analysis of the following nuclear magnetic resonance (NMR) parameters: nuclear Overhauser effect (NOE) data, and C(alpha)H, (13)C(alpha), and (13)C(beta) conformational shifts. From the differences in the ability to adopt beta-hairpin structures in these peptides, we have arrived to the following conclusions: (i) beta-Hairpin population increases with the statistical propensity of residues to occupy each turn position. (ii) The loop length, and in turn, the beta-hairpin type, can be modified as a function of the type of turn favored by the loop sequence. These two conclusions reinforce previous results about the importance of beta-turn sequence in beta-hairpin folding. (iii) Side-chain packing on each face of the beta-sheet may play a major role in beta-hairpin stability; hence simplified analysis in terms of isolated pair interactions and intrinsic beta-sheet propensities is insufficient. (iv) Contributions to beta-hairpin stability of turn and strand sequences are not completely independent. (v) The burial of hydrophobic surface upon beta-hairpin formation that, in turn, depends on side-chain packing also contributes to beta-hairpin stability. (vi) As previously observed, TFE stabilizes beta-hairpin structures, but the extent of the contribution of different factors to beta-hairpin formation is sometimes different in aqueous solution and in 30% TFE.     

Effects of turn residues on beta-hairpin folding--a molecular dynamics study.

Folding of beta-hairpin structures of synthetic peptides has been simulated using the molecular dynamics method with a solvent-referenced potential. Two similar sequences, Ac-MQIFVKS(D)PGKTITLKV-NH(2) and Ac-MQIFVKS(L)PGKTITLKV-NH(2), derived from the N-terminal beta-hairpin of ubiquitin, were used to study the effects of turn residues in beta-hairpin folding. The simulations were carried out for 80 ns at 297 K. With extended initial conformation, the (D)P-containing peptide folded into a stable 2:2 beta-hairpin conformation with a type II' beta-turn at (D)PG. The overall beta-hairpin ratio, calculated by the DSSP algorithm, was 32.6%. With randomly generated initial conformations, the peptide also formed the stable 2:2 beta-hairpin conformation. The interactions among the side chains in the 2:2 beta-hairpin were almost identical to those in the native protein. These interactions reduced the solvation energy upon folding and stabilized the beta-hairpin conformation. Without the solvent effect, the peptide did not fold into stable beta-hairpin structures. The solvent effect is crucial for the formation of the beta-hairpin conformation. The effect of the temperature has also been studied. The (L)P-containing peptide did not fold into a stable beta-hairpin conformation and had a much lower beta-hairpin ratio (16.6%). The( L)P-containing peptide has similar favorable side-chain interactions, but the turn formed by (L)PG does not connect well with the right-handed twist of the beta-strands. For comparison, the isolated N-terminal peptide of ubiquitin, Ac-MQIFVKTLTGKTITLEV-NH(2), was also simulated and its beta-hairpin ratio was low, indicating that the beta-hairpin in the native structure is stabilized by the interaction with the protein environment. These simulation results agreed qualitatively with the available experimental findings.     

The roles of turn formation and cross-strand interactions in fibrillization of peptides derived from the OspA single-layer beta-sheet

We previously demonstrated that a beta-hairpin peptide, termed BH(9-10), derived from a single-layer beta-sheet of Borrelia OspA protein, formed a native-like beta-turn in trifluoroethanol (TFE) solution, and it assembled into amyloid-like fibrils at higher TFE concentrations. This peptide is highly charged, and fibrillization of such a hydrophilic peptide is quite unusual. In this study, we designed a circularly permutated peptide of BH(9-10), termed BH(10-9). When folded into their respective beta-hairpin structures found in OspA, these peptides would have identical cross-strand interactions but different turns connecting the strands. NMR study revealed that BH(10-9) had little propensity to form a turn structure both in aqueous and TFE solutions. At higher TFE concentration, BH(10-9) precipitated with a concomitant alpha-to-beta conformational conversion, in a similar manner to the BH(9-10) fibrillization. However, the BH(10-9) precipitates were nonfibrillar aggregation. The precipitation kinetics of BH(10-9) was exponential, consistent with a first-order molecular assembly reaction, while the fibrillization of BH(9-10) showed sigmoidal kinetics, indicative of a two-step reaction consisting of nucleation and molecular assembly. The correlation between native-like turn formation and fibrillization of our peptide system strongly suggests that BH(9-10) adopts a native-like beta-hairpin conformation in the fibrils. Remarkably, seeding with the preformed BH(10-9) precipitates changed the two-step BH(9-10) fibrillization to a one-step molecular assembly reaction, and disrupted the BH(9-10) fibril structure, indicating interactions between the BH(10-9) aggregates and the BH(9-10) peptide. Our results suggest that, in these peptides, cross-strand interactions are the driving force for molecular assembly, and turn formation limits modes of peptide assembly.     

Micelle-bound conformation of a hairpin-forming peptide: combined NMR and molecular dynamics study

A peptide fragment from a protein hairpin turn region was modified by addition of isoleucine residues to both ends to enhance binding to lipid micelles; the resulting peptide (I(1)-I(2)-C(3)-N(4)-N(5)-P(6)-H(7)-I(8)-I(9)) contains the core sequence I-C-N-N-P-H from an antibody-binding region of hemagglutinin A. Nuclear magnetic resonance (NMR) diffusion measurements indicated partial binding (43-65%) of the peptide to micelles of n-octylglucoside and significantly stronger binding (85%) to dodecylphosphocholine (DPC) micelles. Simulated annealing and conformational analysis using nuclear Overhauser enhancement restraints revealed a type I or III hairpin turn between residues N(5) and I(8) of the DPC-bound peptide. Amide exchange experiments support the possibility that a hydrogen bond forms between N(5) and I(8), stabilizing the turn. In contrast, no discernable structure was observed for the peptide in aqueous solution by either NMR or circular dichroism. Molecular dynamics simulations of DPC micelles and peptide-micelle complexes suggested that the peptide lies flat on the micelle surface and showed rapid rearrangement of the lipids to accommodate the bound peptide. According to a search performed using the basic local alignment search tool (BLAST), the sequences N-P-H-I and N-P-H-V are present as hairpin turns in eight of the nine proteins whose crystal structures were available. The addition of isoleucine residues and the use of lipid micelles to stabilize hairpin conformations equivalent to those found in proteins generates new possibilities for reproducing biologically important hairpin turns from short, linear peptides.     

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.     

The turn sequence directs beta-strand alignment in designed beta-hairpins

A previous NMR investigation of model decapeptides with identical beta-strand sequences and different turn sequences demonstrated that, in these peptide systems, the turn residues played a more predominant role in defining the type of beta-hairpin adopted than cross-strand side-chain interactions. This result needed to be tested in longer beta-hairpin forming peptides, containing more potentially stabilizing cross-strand hydrogen bonds and side-chain interactions that might counterbalance the influence of the turn sequence. In that direction, we report here on the design and 1H NMR conformational study of three beta-hairpin forming pentadecapeptides. The design consists of adding two and three residues at the N- and C-termini, respectively, of the previously studied decapeptides. One of the designed pentadecapeptides includes a potentially stabilizing R-E salt bridge to investigate the influence of this interaction on beta-hairpin stability. We suggest that this peptide self-associates by forming intermolecular salt bridges. The other two pentadecapeptides behave as monomers. A conformational analysis of their 1H NMR spectra reveals that they adopt different types of beta-hairpin structure despite having identical strand sequences. Hence, the beta-turn sequence drives beta-hairpin formation in the investigated pentadecapeptides that adopt beta-hairpins that are longer than the average protein beta-hairpins. These results reinforce our previous suggestion concerning the key role played by the turn sequence in directing the kind of beta-hairpin formed by designed peptides.     

Amino acid determinants of beta-hairpin conformation in erythropoeitin receptor agonist peptides derived from a phage display library

Display of peptide libraries on filamentous phage has led to the identification of peptides of the form X(2-5)CX(2)GPXTWXCX(2-5) (where X is a variable residue) that bind to the extra-cellular portion of the erythropoietin receptor (EPO-R). These peptides adopt beta-hairpin conformations when co-crystallized with EPO-R. Solution NMR studies reveal that the peptide is conformationally heterogeneous in the absence of receptor due to cis-trans isomerization about the Gly-Pro peptide bond. Replacement of the conserved threonine residue with glycine at the turn i+3 position produces a stable beta-hairpin conformation in solution, although this peptide no longer has activity in an EPO-R-dependent cell proliferation assay. A truncated form of the EPO-R-binding peptide (containing the i+3 glycine residue) also forms a highly populated, monomeric beta-hairpin. In contrast, phage-derived peptide antagonists of insulin-like growth factor binding protein 1 (IGFBP-1) have a high level of sequence identity with the truncated EPO-R peptide (eight of 12 residues) yet adopt a turn-alpha-helix conformation in solution. Peptides containing all possible pairwise amino acid substitutions between the EPO-R and IGFBP-1 peptides have been analyzed to assess the degree to which the non-conserved residues stabilize the hairpin or helix conformation. All four residues present in the original sequence are required for maximum population of either the beta-hairpin or alpha-helix conformation, although some substitutions have a more dominant effect. The results demonstrate that, within a given sequence, the observed conformation can be dictated by a small subset of the residues (in this case four out of 12).     

Solution structures of a 30-residue amino-terminal domain of the carp granulin-1 protein and its amino-terminally truncated 3-30 subfragment: implications for the conformational stability of the stack of two beta-hairpins

Carp granulins are members of an emerging class of proteins with a sequence motif encoding a parallel stack of two to four beta-hairpins. The carp granulin-1 protein forms a stack of four beta-hairpins, whereas its amino-terminal fragment appears to adopt a very stable stack of two beta-hairpins in solution. Here we determined a refined three-dimensional structure of this peptide fragment to examine potential conformational changes compared with the full-length protein. The structures were calculated with both a traditional method and a fast semiautomated method using ambiguous NMR distance restraints. The resulting sets of structures are very similar and show that a well-defined stack of two beta-hairpins is retained in the peptide. Conformational rearrangements compensating the loss of the carboxy-terminal subdomain of the native protein are restricted to the carboxy-terminal end of the peptide, the turn connecting the two beta-hairpins, and the Tyr(21) and Tyr(25) aromatic side chains. Further removal of the Val(1) and Ile(2) residues, which are part of the first beta-hairpin and components of two major hydrophobic clusters in the two beta-hairpin structure, results in the loss of the first beta-hairpin. The second beta-hairpin, which is closely associated with the first, retains a similar but somewhat less stable conformation. The invariable presence of the second beta-hairpin and the dependence of its stability on the first beta-hairpin suggest that the stack of two beta-hairpins may be an evolutionary conserved and autonomous folding unit. In addition, the high conformational stability makes the stack of two beta-hairpins an attractive scaffold for the development of peptide-based drug candidates.     

Beta-hairpin folding mechanism of a nine-residue peptide revealed from molecular dynamics simulations in explicit water

The beta-hairpin fold mechanism of a nine-residue peptide, which is modified from the beta-hairpin of alpha-amylase inhibitor tendamistat (residues 15-23), is studied through direct folding simulations in explicit water at native folding conditions. Three 300-nanosecond self-guided molecular dynamics (SGMD) simulations have revealed a series of beta-hairpin folding events. During these simulations, the peptide folds repeatedly into a major cluster of beta-hairpin structures, which agree well with nuclear magnetic resonance experimental observations. This major cluster is found to have the minimum conformational free energy among all sampled conformations. This peptide also folds into many other beta-hairpin structures, which represent some local free energy minimum states. In the unfolded state, the N-terminal residues of the peptide, Tyr-1, Gln-2, and Asn-3, have a confined conformational distribution. This confinement makes beta-hairpin the only energetically favored structure to fold. The unfolded state of this peptide is populated with conformations with non-native intrapeptide interactions. This peptide goes through fully hydrated conformations to eliminate non-native interactions before folding into a beta-hairpin. The folding of a beta-hairpin starts with side-chain interactions, which bring two strands together to form interstrand hydrogen bonds. The unfolding of the beta-hairpin is not simply the reverse of the folding process. Comparing unfolding simulations using MD and SGMD methods demonstrate that SGMD simulations can qualitatively reproduce the kinetics of the peptide system.     

Solution conformations of two flexible cyclic pentapeptides: cyclo(Gly-Pro-D-Phe-Gly-Ala) and cyclo(Gly-Pro-D-Phe-Gly-Val).

In an effort to explore the residue preferences in three-residue reverse turns (so-called gamma-turns), two cyclic pentapeptides--cyclo(Gly1-Pro2-D-Phe3-Gly4-Ala5) (I) and cyclo(Gly1-Pro2-D-Phe3-Gly4-Val5) (II)--have been synthesized and analyzed by nmr. It was anticipated that the Gly-Pro-D-Phe-Gly portions of these molecules would favor a beta-turn conformation, leaving the remainder of the molecule to adopt a gamma turn, as seen in several previously studied model cyclic pentapeptides. The nmr data for both peptides in CDCl3 (5% DMSO-d6) and in neat DMSO-d6 indicate that the most populated conformation contains a distorted beta turn around Pro2-D-Phe3, which includes a gamma turn around D-Phe3. The distortion in the beta turn does not impede the formation of an inverse gamma turn around residue 5, and indeed, this conformation is observed in both peptides. Both the alanine and the bulkier valine residues are therefore found to be compatible with an inverse gamma turn. Molecular dynamics simulations on the title peptides are reported in the following paper. These simulations indicate that there is conformational flexibility around the D-Phe3-Gly4 peptide bond, which enables the formation of the gamma turn around D-Phe3. The third paper in this series explores the impact of a micellar environment on conformational equilibria in II.     

Stability of monomeric Cro variants: Isoenergetic transformation of a type I' to a type II' beta-hairpin by single amino acid replacements

The thermodynamic stabilities of three monomeric variants of the bacteriophage lambda Cro repressor that differ only in the sequence of two amino acids at the apex of an engineered beta-hairpin have been determined. The sequences of the turns are EVK-XX-EVK, where the two central residues are DG, GG, and GT, respectively. Standard-state unfolding free energies, determined from circular dichroism measurements as a function of urea concentration, range from 2.4 to 2.7 kcal/mole, while those determined from guanidine hydrochloride range from 2.8 to 3.3 kcal/mole for the three proteins. Thermal denaturation yields van't Hoff unfolding enthalpies of 36 to 40 kcal /mole at midpoint temperatures in the range of 53 to 58 degrees C. Extrapolation of the thermal denaturation free energies with heat capacities of 400 to 600 cal/mole deg gives good agreement with the parameters determined in denaturant titrations. As predicted from statistical surveys of amino acid replacements in beta-hairpins, energetic barriers to transformation from a type I' turn (DG) to a type II' turn (GT) can be quite small.     

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.     

Solution structure of the calcium channel antagonist omega-conotoxin GVIA.

The three-dimensional solution structure is reported for omega-conotoxin GVIA, which is a potent inhibitor of presynaptic calcium channels in vertebrate neuromuscular junctions. Structures were generated by a hybrid distance geometry and restrained molecular dynamics approach using interproton distance, torsion angle, and hydrogen-bonding constraints derived from 1H NMR data. Conformations of GVIA with low constraint violations converged to a common peptide fold. The secondary structure in the peptide is an antiparallel triple-stranded beta-sheet containing a beta-hairpin and three tight turns. The NMR data are consistent with the region of the peptide from residues S9 to C16 being more dynamic than the rest of the peptide. The peptide has an amphiphilic structure with a positively charged hydrophilic side and an opposite side that contains a small hydrophobic region. Residues that are thought to be important in binding and function are located on the hydrophilic face of the peptide.     

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.     

Nanosecond time scale folding dynamics of a pentapeptide in water

Reverse turns, four-residue sections of polypeptides where the chain changes direction by about 180 degrees, are thought to be important protein folding initiation structures. However, the time scale and mechanism for their formation have yet to be determined experimentally. To develop a microscopic picture of the formation of protein folding initiation structures, we have carried out a pair of 2.2-ns molecular dynamics simulations of Tyr-Pro-Gly-Asp-Val, a peptide which is known to form a high population of reverse turns in water. In the first simulation, which was started with the peptide in an ideal type II reverse turn involving the first four residues, the turn unfolded after about 1.4 ns. After about 0.6 ns in the second simulation, which was started with the peptide in a fully extended conformation, the peptide folded into a type II turn which had a transient existence before unfolding. The peptide remained unfolded for another 0.9 ns before folding into a type I turn involving the last four residues. The type I turn lasted for about 0.2 ns before unfolding. Thus, these simulations showed that protein folding initiation structures can form and dissolve on the nanosecond time scale. Furthermore, the atomic-level detail of the simulations allowed us to identify some of the interactions which can stabilize the folded structures. The type II turns were stabilized by either a salt bridge between the terminal groups or a backbone-C-terminal group hydrogen bond, and the type I turns were stabilized by a hydrophobic interaction between the proline and valine-side chains.     

Dissecting the stability of a beta-hairpin peptide that folds in water: NMR and molecular dynamics analysis of the beta-turn and beta-strand contributions to folding.

NMR studies of the folding and conformational properties of a beta-hairpin peptide, several peptide fragments of the hairpin, and sequence-modified analogues, have enabled the various contributions to beta-hairpin stability in water to be dissected. Temperature and pH-induced unfolding studies indicate that the folding-unfolding equilibrium approximates to a two-state model. The hairpin is highly resistant to denaturation and is still significantly folded in 7 M urea at 298 K. Thermodynamic analysis shows the hairpin to fold in water with a significant change in heat capacity, however, DeltaCp degrees in 7 M urea is reduced. V/Y-->A mutations on one strand of the hairpin reduce folding to <10 %, consistent with a hydrophobic stabilisation model. We show that in a truncated peptide (residues 6-16) lacking the hydrophobic residues on one beta-strand, the type I' Asn-Gly turn in the sequence SINGKK is significantly populated in water in the absence of interstrand hydrophobic contacts. Unrestrained molecular dynamics simulations of unfolding, using an explicit solvation model, show that the conformation of the NG turn persists for longer than the AG analogue, which has a much lower propensity for type I' turn formation from a data base analysis of preferred turns. The origin of the high stability of the Asn-Gly turn is not entirely clear; data base analysis of 66 NG turns, together with molecular dynamics simulations, reveals no participation of the Asn side-chain in turn-stabilising interactions with the peptide backbone. However, hydration analysis of the molecular dynamics simulations reveals a pocket of "high density" water bridging between the Asn side-chain and peptide main-chain that suggests solvent-mediated interactions may play an important role in modulating phi,psi propensities in the NG turn region.     

Native topology or specific interactions: what is more important for protein folding?

Fifty-five molecular dynamics runs of two three-stranded antiparallel beta-sheet peptides were performed to investigate the relative importance of amino acid sequence and native topology. The two peptides consist of 20 residues each and have a sequence identity of 15 %. One peptide has Gly-Ser (GS) at both turns, while the other has d-Pro-Gly ((D)PG). The simulations successfully reproduce the NMR solution conformations, irrespective of the starting structure. The large number of folding events sampled along the trajectories at 360 K (total simulation time of about 5 micros) yield a projection of the free-energy landscape onto two significant progress variables. The two peptides have compact denatured states, similar free-energy surfaces, and folding pathways that involve the formation of a beta-hairpin followed by consolidation of the unstructured strand. For the GS peptide, there are 33 folding events that start by the formation of the 2-3 beta-hairpin and 17 with first the 1-2 beta-hairpin. For the (D)PG peptide, the statistical predominance is opposite, 16 and 47 folding events start from the 2-3 beta-hairpin and the 1-2 beta-hairpin, respectively. These simulation results indicate that the overall shape of the free-energy surface is defined primarily by the native-state topology, in agreement with an ever-increasing amount of experimental and theoretical evidence, while the amino acid sequence determines the statistically predominant order of the events.