Stabilization of the N-terminal beta-hairpin of ubiquitin by a terminal hydrophobic cluster

Study of model beta-hairpin peptides allows for better understanding of the factors involved in the formation of beta-sheet secondary structure in proteins. It is known that turn sequence, sidechain-sidechain interactions, interstrand hydrogen bonding, and beta-sheet propensity of residues are all important for beta-hairpin stability in aqueous solution. However, interactions of the sidechains of the terminal residues of hairpins are thought to contribute little to overall hairpin stability since these residues are typically frayed. Here, the authors report a stabilizing hydrophobic cluster of residues at the termini of the naturally occurring excised N-terminal beta-hairpin of Ubiquitin that folds autonomously in aqueous solution. Our data show that deletion of Met1 and Val17 from this hairpin destabilized the folded state in both aqueous solution and in aqueous-methanol solutions. These results suggest that interactions of terminal residues which are usually frayed can nonetheless contribute significantly to overall stability of beta-hairpin.     

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.     

Model systems for beta-hairpins and beta-sheets

beta-Sheets and alpha-helices are the two principal secondary structures in proteins. However, our understanding of beta-sheet structure lags behind that of alpha-helices, largely because, until recently, there was no model system to study the beta-sheet secondary structure in isolation. With the development of well-folded beta-hairpins, this is changing rapidly. Recent advances include: increased understanding of the relative contributions of turn, strand and sidechain interactions to beta-hairpin and beta-sheet stability, with the role of aromatic residues as a common subtheme; experimental and theoretical kinetic and thermodynamic studies of beta-hairpin and beta-sheet folding; de novo protein design, including all-beta structures, mixed alpha/beta motifs and switchable systems; and the creation of functional beta-hairpins.     

Interplay between hydrophobic cluster and loop propensity in beta-hairpin formation

Autonomously folding beta-hairpins have recently emerged as powerful tools for elucidating the origins of antiparallel beta-sheet folding preferences. Analysis of such model systems has suggested four potential sources of beta-sheet stability: (1) the conformational propensity of the loop segment that connects adjacent strands; (2) favorable contacts between side-chains on adjacent strands; (3) interstrand hydrogen bonds; and (4) the intrinsic beta-sheet propensities of the strand residues. We describe the design and analysis of a series of isomeric 20 residue peptides in which factors (1)-(4) are identical. Differences in beta-hairpin formation within this series demonstrate that these four factors, individually, are not sufficient to explain beta-sheet stability. In agreement with the prediction of a simple statistical mechanical model for beta-hairpin formation, our results show that the separation between the loop segment and an interstrand cluster of hydrophobic side-chains strongly influences beta-hairpin size and stability, with a smaller separation leading to greater stability.     

Elongation of the BH8 β-hairpin peptide: Electrostatic interactions in β-hairpin formation and stability

An elongated version of the de novo designed beta-hairpin peptide, BH8, has allowed us to gain insight into the role of electrostatic interactions in beta-hairpin stability. A Lys-Glu electrostatic pair has been introduced by adding a residue at the beginning and at the end of the N-terminal and C-terminal strands, respectively, of the beta-hairpin structure, in both orientations. The two resulting peptides and controls having Ala residues at these positions and different combinations of Ala with Lys, or Glu residues, have been analyzed by nuclear magnetic resonance (NMR), under different pH and ionic strength conditions. All of the NMR parameters, in particular the conformational shift analysis of Calpha protons and the coupling constants, (3)J(HNalpha), correlate well and the population estimates are in reasonable agreement among the different methods used. In the most structured peptides, we find an extension of the beta-hairpin structure comprising the two extra residues. Analysis of the pH and salt dependence shows that ionic pairs contribute to beta-hairpin stability. The interaction is electrostatic in nature and can be screened by salt. There is also an important salt-independent contribution of negatively charged groups to the stability of this family of beta-hairpin peptides.     

Minimal model for studying prion-like folding pathways

The Monte Carlo technique is used to simulate the energy landscape and the folding kinetics of a minimal prion-like protein model. We show that the competition between hydrogen-bonding and hydrophobic interactions yields two energetically favored secondary structures, an alpha-helix and a beta-hairpin. Folding simulations indicate that the probability of reaching the alpha-helix form from a denatured random conformation is much higher than the probability of reaching the beta-sheet form, even though the beta-sheet has a lower energy. The existence of a lower energy beta-sheet state gives the possibility for the normal alpha-helix structure to take a structural transformation into the beta-sheet structure under external influences.     

Synthesis, folding, and structure of the beta-turn mimic modified B1 domain of streptococcal protein G

The mechanism of beta-sheet formation remains a fundamental issue in our understanding of the protein folding process, but is hampered by the often encountered kinetic competition between folding and aggregation. The role of local versus nonlocal interactions has been probed traditionally by mutagenesis of both turn and strand residues. Recently, rigid organic molecules that impose a correct chain reversal have been introduced in several small peptides to isolate the importance of the long-range interactions. Here, we present the incorporation of a well-studied beta-turn mimic, designated as the dibenzofuran-based (DBF) amino acid, in the B1 domain of streptococcal protein G (B1G), and compare our results with those obtained upon insertion of the same mimic into the N-terminal beta-hairpin of B1G (O Melnyk et al., 1998, Lett Pept Sci 5:147-150). The DBF-B1G domain conserves the structure and the functional and thermodynamical properties of the native protein, whereas the modified peptide does not adopt a native-like conformation. The nature of the DBF flanking residues in the modified B1G domain prevents the beta-turn mimic from acting as a strong beta-sheet nucleator, which reinforces the idea that the native beta-hairpin formation is not driven by the beta-turn formation, but by tertiary interactions.     

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.     

NMR solution structure, stability, and interaction of the recombinant bovine fibrinogen alphaC-domain fragment

According to the existing hypothesis, in fibrinogen, the COOH-terminal portions of two Aalpha chains are folded into compact alphaC-domains that interact intramolecularly with each other and with the central region of the molecule; in fibrin, the alphaC-domains switch to an intermolecular interaction resulting in alphaC-polymers. In agreement, our recent NMR study identified within the bovine fibrinogen Aalpha374-538 alphaC-domain fragment an ordered compact structure including a beta-hairpin restricted at the base by a 423-453 disulfide linkage. To establish the complete structure of the alphaC-domain and to further test the hypothesis, we expressed a shorter alphaC-fragment, Aalpha406-483, and performed detailed analysis of its structure, stability, and interactions. NMR experiments on the Aalpha406-483 fragment identified a second loose beta-hairpin formed by residues 459-476, yielding a structure consisting of an intrinsically unstable mixed parallel/antiparallel beta-sheet. Size-exclusion chromatography and sedimentation velocity experiments revealed that the Aalpha406-483 fragment forms soluble oligomers whose fraction increases with an increase in concentration. This was confirmed by sedimentation equilibrium analysis, which also revealed that the addition of each monomer to an assembling alphaC-oligomer substantially increases its stabilizing free energy. In agreement, unfolding experiments monitored by CD established that oligomerization of Aalpha406-483 results in increased thermal stability. Altogether, these experiments establish the complete NMR solution structure of the Aalpha406-483 alphaC-domain fragment, provide direct evidence for the intra- and intermolecular interactions between the alphaC-domains, and confirm that these interactions are thermodynamically driven.     

The design of linear peptides that fold as monomeric beta-sheet structures.

Current knowledge about the determinants of beta-sheet formation has been notably improved by the structural and kinetic analysis of model peptides, by mutagenesis experiments in proteins and by the statistical analysis of the protein structure database (Protein Data Bank; PDB). In the past year, several peptides comprising natural and non-natural amino acids have been designed to fold as monomeric three-stranded beta-sheets. In all these cases, the design strategy has involved both the statistical analysis of the protein structure database and empirical information obtained in model beta-hairpin systems and in proteins. Only in one case was rotamer analysis performed to check for the compatibility of the sidechain packing. It is foreseeable that, in future designs, algorithms exploring the sequence and conformational space will be employed. For the design of small proteins (less than 30 amino acids), questions remain about the demonstration of two-state behavior, the formation of a well-defined network of mainchain hydrogen bonds and the quantification of the structured populations.     

A proposed structural model for amyloid fibril elongation: domain swapping forms an interdigitating beta-structure polymer

We propose a model illustrating how proteins, which differ in their overall sequences and structures, can form the propagating, twisted beta-sheet conformations, characteristic of amyloids. Some cases of amyloid formation can be explained through a "domain swapping" event, where the swapped segment is either a beta-hairpin or an unstable conformation which can partially unfold and assume a beta-hairpin structure. As in domain swapping, here the swapped beta-hairpin is at the edge of the structure, has few (if any) salt bridges and hydrogen bonds connecting it to the remainder of the structure and variable extents of buried non-polar surface areas. Additionally, in both cases the swapped piece constitutes a transient "building block" of the structure, with a high population time. Whereas in domain swapping the swapped fragment has been shown to be an alpha-helix, loop, strand or an entire domain, but so far not a beta-hairpin, despite the large number of cases in which it was already detected, here swapping may involve such a structural motif. We show how the swapping of beta-hairpins would form an interdigitated, twisted beta-sheet conformation, explaining the remarkable high stability of the protofibril in vitro. Such a swapping mechanism is attractive as it involves a universal mechanism in proteins, critical for their function, namely hinge-bending motions. Our proposal is consistent with structural superpositioning of mutational variants. While the overall r.m.s.d.s of the wild-type and mutants are small, the proposed hinge-bending region consistently shows larger deviations. These larger deviations illustrate that this region is more prone to respond to the mutational changes, regardless of their location in the sequence or in the structure. Nevertheless, above all, we stress that this proposition is hypothetical, since it is based on assumptions lacking definitive experimental support.     

Cross-strand side-chain interactions versus turn conformation in beta-hairpins.

A series of designed peptides has been analyzed by 1H-NMR spectroscopy in order to investigate the influence of cross-strand side-chain interactions in beta-hairpin formation. The peptides differ in the N-terminal residues of a previously designed linear decapeptide that folds in aqueous solution into two interconverting beta-hairpin conformations, one with a type I turn (beta-hairpin 4:4) and the other with a type I + G1 beta-bulge turn (beta-hairpin 3:5). Analysis of the conformational behavior of the peptides studied here demonstrates three favorable and two unfavorable cross-strand side-chain interactions for beta-hairpin formation. These results are in agreement with statistical data on side-chain interactions in protein beta-sheets. All the peptides in this study form significant populations of the beta-hairpin 3:5, but only some of them also adopt the beta-hairpin 4:4. The formation of beta-hairpin 4:4 requires the presence of at least two favorable cross-strand interactions, whereas beta-hairpin 3:5 seems to be less susceptible to side-chain interactions. A protein database analysis of beta-hairpins 3:5 and beta-hairpins 4:4 indicates that the former occur more frequently than the latter. In both peptides and proteins, beta-hairpins 3:5 have a larger right-handed twist than beta-hairpins 4:4, so that a factor contributing to the higher stability of beta-hairpin 3:5 relative to beta-hairpin 4:4 is due to an appropriate backbone conformation of the type I + G1 beta-bulge turn toward the right-handed twist usually observed in protein beta-sheets. In contrast, as suggested previously, backbone geometry of the type I turn is not adequate for the right-handed twist. Because analysis of buried hydrophobic surface areas on protein beta-hairpins reveals that beta-hairpins 3:5 bury more hydrophobic surface area than beta-hairpins 4:4, we suggest that the right-handed twist observed in beta-hairpin 3:5 allows a better packing of side chains and that this may also contribute to its higher intrinsic stability.     

The mechanism of beta-hairpin formation

Beta-hairpins constitute an important class of connecting protein secondary structures. Several groups have postulated that such structures form early in the folding process and serve to nucleate the formation of extended beta-sheet structures. Despite the importance of beta-hairpins in protein folding, little is known about the mechanism of formation of these structures. While it is well established that there is a complex interplay between the stability of a beta-hairpin and loop conformational propensity, loop length, and the formation of stabilizing cross-strand interactions (H-bonds and hydrophobic interactions), the influence of these factors on the folding rate is poorly understood. Peptide models provide a simple framework for exploring the molecular details of the formation of beta-hairpin structures. We have explored the fundamental processes of folding in two linear peptides that form beta-hairpin structures, having a stabilizing hydrophobic cluster connected by loops of differing lengths. This approach allows us to evaluate existing models of the mechanism of beta-hairpin formation. We find a substantial acceleration of the folding rate when the connecting loop is made shorter (i.e., the hydrophobic cluster is moved closer to the turn). Analysis of the folding kinetics of these two peptides reveals that this acceleration is a direct consequence of the reduced entropic cost of the smaller loop search.     

Mutational analysis of the folding transition state of the C-terminal domain of ribosomal protein L9: a protein with an unusual beta-sheet topology

The C-terminal domain of ribosomal protein L9 (CTL9) is a 92-residue alpha-beta protein which contains an unusual three-stranded mixed parallel and antiparallel beta-sheet. The protein folds in a two-state fashion, and the folding rate is slow. It is thought that the slow folding may be caused by the necessity of forming this unusual beta-sheet architecture in the transition state for folding. This hypothesis makes CTL9 an interesting target for folding studies. The transition state for the folding of CTL9 was characterized by phi-value analysis. The folding of a set of hydrophobic core mutants was analyzed together with a set of truncation mutants. The results revealed a few positions with high phi-values (> or = 0.5), notably, V131, L133, H134, V137, and L141. All of these residues were found in the beta-hairpin region, indicating that the formation of this structure is likely to be the rate-limiting step in the folding of CTL9. One face of the beta-hairpin docks against the N-terminal helix. Analysis of truncation mutants of this helix confirmed its importance in folding. Mutations at other sites in the protein gave small phi-values, despite the fact that some of them had major effects on stability. The analysis indicates that formation of the antiparallel hairpin is critical and its interactions with the first helix are also important. Thus, the slow folding is not a consequence of the need to fully form the unusual three-stranded beta-sheet in the transition state. Analysis of the urea dependence of the folding rates indicates that mutations modulate the unfolded state. The folding of CTL9 is broadly consistent with the nucleation-condensation model of protein folding.     

Structural analysis of WW domains and design of a WW prototype

Two new NMR structures of WW domains, the mouse formin binding protein and a putative 84.5 kDa protein from Saccharomyces cerevisiae, show that this domain, only 35 amino acids in length, defines the smallest monomeric triple-stranded antiparallel beta-sheet protein domain that is stable in the absence of disulfide bonds, tightly bound ions or ligands. The structural roles of conserved residues have been studied using site-directed mutagenesis of both wild type domains. Crucial interactions responsible for the stability of the WW structure have been identified. Based on a network of highly conserved long range interactions across the beta-sheet structure that supports the WW fold and on a systematic analysis of conserved residues in the WW family, we have designed a folded prototype WW sequence.     

NMR study and molecular dynamics simulations of optimized beta-hairpin fragments of protein G

The stability and structure of several beta-hairpin peptide variants derived from the C-terminus of the B1 domain of protein G were investigated by a number of experimental and computational techniques. Our analysis shows that the structure and stability of this hairpin can be greatly affected by one or a few simple mutations. For example, removing an unfavorable charge near the N-terminus of the peptide (Glu42 to Gln or Thr) or optimization of the N-terminal charge-charge interactions (Gly41 to Lys) both stabilize the peptide, even in water. Furthermore, a simple replacement of a charged residue in the turn (Asp47 to Ala) changes the beta-turn conformation. Finally, we show that the effects of combining these single mutations are additive, suggesting that independent stabilizing interactions can be isolated and evaluated in a simple model system. Our results indicate that the structure and stability of this beta-hairpin peptide can be modulated in numerous ways and thus contributes toward a more complete understanding of this important model beta-hairpin as well as to the folding and stability of larger peptides and proteins.     

Mechanism for the alpha-helix to beta-hairpin transition

The aggregation of alpha-helix-rich proteins into beta-sheet-rich amyloid fibrils is associated with fatal diseases, such as Alzheimer's disease and prion disease. During an aggregation process, protein secondary structure elements-alpha-helices-undergo conformational changes to beta-sheets. The fact that proteins with different sequences and structures undergo a similar transition on aggregation suggests that the sequence nonspecific hydrogen bond interaction among protein backbones is an important factor. We perform molecular dynamics simulations of a polyalanine model, which is an alpha-helix in its native state and observe a metastable beta-hairpin intermediate. Although a beta-hairpin has larger potential energy than an alpha-helix, the entropy of a beta-hairpin is larger because of fewer constraints imposed by the hydrogen bonds. In the vicinity of the transition temperature, we observe the interconversion of the alpha-helix and beta-sheet states via a random coil state. We also study the effect of the environment by varying the relative strength of side-chain interactions for a designed peptide-an alpha-helix in its native state. For a certain range of side-chain interaction strengths, we find that the intermediate beta-hairpin state is destabilized and even disappears, suggesting an important role of the environment in the aggregation propensity of a peptide.     

Thermodynamic and kinetic characterization of a beta-hairpin peptide in solution: an extended phase space sampling by molecular dynamics simulations in explicit water

The folding of the amyloidogenic H1 peptide MKHMAGAAAAGAVV taken from the syrian hamster prion protein is explored in explicit aqueous solution at 300 K using long time scale all-atom molecular dynamics simulations for a total simulation time of 1.1 mus. The system, initially modeled as an alpha-helix, preferentially adopts a beta-hairpin structure and several unfolding/refolding events are observed, yielding a very short average beta-hairpin folding time of approximately 200 ns. The long time scale accessed by our simulations and the reversibility of the folding allow to properly explore the configurational space of the peptide in solution. The free energy profile, as a function of the principal components (essential eigenvectors) of motion, describing the main conformational transitions, shows the characteristic features of a funneled landscape, with a downhill surface toward the beta-hairpin folded basin. However, the analysis of the peptide thermodynamic stability, reveals that the beta-hairpin in solution is rather unstable. These results are in good agreement with several experimental evidences, according to which the isolated H1 peptide adopts very rapidly in water beta-sheet structure, leading to amyloid fibril precipitates [Nguyen et al., Biochemistry 1995;34:4186-4192; Inouye et al., J Struct Biol 1998;122:247-255]. Moreover, in this article we also characterize the diffusion behavior in conformational space, investigating its relations with folding/unfolding conditions.     

Structural malleability of plasticins: preorganized conformations in solution and relevance for antimicrobial activity

Plasticins (23 long-residue glycine-leucine-rich dermaseptin-related peptides produced by the skin of South American hylids) have very similar amino acid sequences, hydrophobicities, and amphipathicities, but differ in their membrane-damaging properties and structurations (i.e. destabilized helix states, beta-hairpin, beta-sheet, and disordered states) at anionic and zwitterionic membrane interfaces. Structural malleability of plasticins in aqueous solutions together with parameters that may govern their ability to fold within beta-hairpin like structures were analyzed through circular dichroism and FTIR spectroscopic studies completed by molecular dynamics simulations in polar mimetic media. The goal of this study was to probe to which extent pre-existent peptide conformations, i.e. intrinsic "conformational landscape", may be responsible for variability in bioactive conformation and antimicrobial/hemolytic mechanisms of action of these peptides in relation with their various membrane disturbing properties. All plasticins present a turn region that does not always result in folding into a beta-hairpin shaped conformation. Residue at position 8 plays a major role in initiating the folding, while position 12 is not critical. Conformational stability has no major impact on antimicrobial efficacy. However, preformed beta-hairpin in solution may act as a conformational lock that prevents switch to alpha-helical structure. This lock lowers the antimicrobial efficiency and explains subtle differences in potencies of the most active antimicrobial plasticins.     

Free energy surfaces of beta-hairpin and alpha-helical peptides generated by replica exchange molecular dynamics with the AGBNP implicit solvent model

We have studied the potential of mean force of two peptides, one known to adopt a beta-hairpin and the other an alpha-helical conformation in solution. These peptides are, respectively, residues 41-56 of the C-terminus (GEWTYDDATKTFTVTE) of the B1 domain of protein G and the 13 residue C-peptide (KETAAAKFERQHM) of ribonuclease A. Extensive canonical ensemble sampling has been performed using a parallel replica exchange method. The effective potential employed in this work consists of the OPLS all-atom force field (OPLS-AA) and an analytical generalized Born (AGB) implicit solvent model including a novel nonpolar solvation free energy estimator (NP). An additional dielectric screening parameter has been incorporated into the AGBNP model. In the case of the beta-hairpin, the nonpolar solvation free energy estimator provides the necessary effective interactions for the collapse of the hydrophobic core (W43, Y45, F52, and V54), which the more commonly used surface-area-dependent nonpolar model does not provide. For both the beta-hairpin and the alpha-helix, increased dielectric screening reduces the stability of incorrectly formed salt bridges, which tend to disrupt the formation of the hairpin and helix, respectively. The fraction of beta-hairpin and alpha-helix content we obtained using the AGBNP model agrees well with experimental results. The thermodynamic stability of the beta-hairpin from protein G and the alpha-helical C-peptide from ribonuclease A as modeled with the OPLS-AA/AGBNP effective potential reflects the balance between the nonpolar effective potential terms, which drive compaction, and the polar and hydrogen bonding terms, which promote secondary structure formation.