Solution conformation and amyloid-like fibril formation of a polar peptide derived from a beta-hairpin in the OspA single-layer beta-sheet

A 23-residue peptide termed BH(9-10) was designed based on a beta-hairpin segment of the single-layer beta-sheet region of Borrelia OspA protein. The peptide contains a large number of charged amino acid residues, and it does not follow the amphipathic pattern that is commonly found in natural beta-sheets. In aqueous solution, the peptide was highly soluble and flexible, with a propensity to form a non-native beta-turn. Trifluoroethanol (TFE) stabilized a native-like beta-turn in BH(9-10). TFE also decreased the level of solubility of the peptide, resulting in peptide precipitation. The precipitation process accompanied a conformational conversion to a beta-sheet structure, as judged with circular dichroism spectroscopy. The precipitate was found to be fibrils similar to those associated with human amyloid diseases. The fibrillization kinetics depended on peptide and TFE concentrations, and had a nucleation step followed by an assembly step. The fibrillization was reversible, and the dissociation reaction involved two phases. TFE appears to induce the fibrils by stabilizing a beta-sheet conformation of the peptide that optimally satisfies hydrogen bonding and electrostatic complementarity. This TFE-induced fibrillization is quite unusual, because most amyloidogenic peptides form fibrils in aqueous solution and TFE disrupts these fibrils. Nevertheless, the BH(9-10) fibrils have similar structure to other fibrils, supporting the emerging idea that polypeptides possess an intrinsic ability to form amyloid-like fibrils. The high level of solubility of BH(9-10), the ability to precisely control fibril formation and dissociation, and the high-resolution structure of the same sequence in the beta-hairpin conformation in the OspA protein provide a tractable experimental system for studying the fibril formation mechanism.     

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.     

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.     

Energy landscape of a peptide consisting of alpha-helix, 3(10)-helix, beta-turn, beta-hairpin, and other disordered conformations

The energy landscape of a peptide [Ace-Lys-Gln-Cys-Arg-Glu-Arg-Ala-Nme] in explicit water was studied with a multicanonical molecular dynamics simulation, and the AMBER parm96 force field was used for the energy calculation. The peptide was taken from the recognition helix of the DNA-binding protein, c-MYB: A rugged energy landscape was obtained, in which the random-coil conformations were dominant at room temperature. The CD spectra of the synthesized peptide revealed that it is in the random state at room temperature. However, the 300 K canonical ensemble, Q(300K), contained alpha-helix, 3(10)-helix, beta-turn, and beta-hairpin structures with small but notable probabilities of existence. The complete alpha-helix, imperfect alpha-helix, and random-coil conformations were separated from one another in the conformational space. This means that the peptide must overcome energy barriers to form the alpha-helix. The overcoming process may correspond to the hydrogen-bond rearrangements from peptide-water to peptide-peptide interactions. The beta-turn, imperfect 3(10)-helix, and beta-hairpin structures, among which there are no energy barriers at 300 K, were embedded in the ensemble of the random-coil conformations. Two types of beta-hairpin with different beta-turn regions were observed in Q(300K). The two beta-hairpin structures may have different mechanisms for the beta-hairpin formation. The current study proposes a scheme that the random state of this peptide consists of both ordered and disordered conformations. In contrast, the energy landscape obtained from the parm94 force field was funnel like, in which the peptide formed the helical conformation at room temperature and random coil at high temperature.     

Sequence dependence of beta-hairpin structure: comparison of a salt bridge and an aromatic interaction

A comparison of the contributions and position dependence of cross-strand electrostatic and aromatic side-chain interactions to beta-sheet stability has been performed by using nuclear magnetic resonance in a well-folded beta-hairpin peptide of the general sequence XRTVXVdPGOXITQX. Phe-Phe and Glu-Lys pairs were varied at the internal and terminal non-hydrogen-bonded position, and the resulting stability was measured by the effects on alpha-hydrogen and aromatic hydrogen chemical shifts. It was determined that the introduction of a Phe-Phe pair resulted in a more folded peptide, regardless of position, and a more tightly folded core. Substitution of the Glu-Lys pair at the internal position results in a less folded peptide and increased fraying at the terminal residues. Upfield shifting of the aromatic hydrogens provided evidence for an edge-face aromatic interaction, regardless of position of the Phe-Phe pair. In peptides with two Phe-Phe pairs, substitution with Glu-Lys at either position resulted in a weakening of the aromatic interaction and a subsequent decrease in peptide stability. Thermal denaturation of the peptides containing Phe-Phe indicates that the aromatic interaction is enthalpically favored, whereas the folding of hairpins with cross-strand Glu-Lys pairs was less enthalpically favorable but entropically more favorable.     

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.     

Reinvestigation of the proposed folding and self-association of the Neuropeptide Head Activator

The Neuropeptide Head Activator (HA), pGlu-Pro-Pro-Gly-Gly-Ser-Lys-Val-Ile-Leu-Phe (pGlu is pyroglutamic acid), is involved in head-specific growth and differentiation processes in the freshwater coelenterate Hydra attenuata. Peptides of identical sequence have also been isolated from higher-organism tissues such as human and bovine hypothalamus. Early studies by molecular sieve chromatography suggested that HA dimerizes with high affinity (K(d) approximately 1 nM). This dimerization was proposed to occur via antiparallel beta-sheet formation between the Lys(7)-Phe(11) segments in each HA molecule. We conducted biophysical studies on synthetic HA in order to gain insight into its structure and aggregation tendencies. We found by analytical ultracentrifugation that HA is monomeric at low millimolar concentrations. Studies by (1)H-NMR revealed that HA did not adopt any significant secondary structure in solution. We found no NOEs that would support the proposed dimer structure. We probed the propensity of the Lys(7)-Phe(11) fragment to form antiparallel beta-sheet by designing peptides in which two such fragments are joined by a two-residue linker. These peptides were intended to form stable beta-hairpin structures with cross-strand interactions that mimic those of the proposed HA dimer interface. We found that the HA-derived fragments may be induced to form intramolecular beta-sheet, albeit only weakly, when linked by the highly beta-hairpin-promoting D-Pro-Gly turn, but not when linked by the more flexible Gly-Gly unit. These findings suggest that the postulated mode of HA dimerization and the proposed propensity of the molecule to form discrete aggregates with high affinity are incorrect.     

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.     

A cross-strand Trp Trp pair stabilizes the hPin1 WW domain at the expense of function

Using the human Pin1 WW domain (hPin1 WW), we show that replacement of two nearest neighbor non-hydrogen-bonded residues on adjacent beta-strands with tryptophan (Trp) residues increases beta-sheet thermodynamic stability by 4.8 kJ mol(-1) at physiological temperature. One-dimensional NMR studies confirmed that introduction of the Trp-Trp pair does not globally perturb the structure of the triple-stranded beta-sheet, while circular dichroism studies suggest that the engineered cross-strand Trp-Trp pair adopts a side-chain conformation similar to that first reported for a designed "Trp-zipper" beta-hairpin peptide, wherein the indole side chains stack perpendicular to each other. Even though the mutated side chains in wild-type hPin1 WW are not conserved among WW domains and compose the beta-sheet surface opposite to that responsible for ligand binding, introduction of the cross-strand Trp-Trp pair effectively eliminates hPin1 WW function as assessed by the loss of binding affinity toward a natural peptide ligand. Maximizing both thermodynamic stability and the domain function of hPin1 WW by the above mentioned approach appears to be difficult, analogous to the situation with loop 1 optimization explored previously. That introduction of a non-hydrogen-bonded cross-strand Trp-Trp pair within the hPin1 WW domain eliminates function may provide a rationale for why this energetically favorable pairwise interaction has not yet been identified in WW domains or any other biologically evolved protein with known three-dimensional structure.     

Turn stability in beta-hairpin peptides: Investigation of peptides containing 3:5 type I G1 bulge turns

The turn-forming ability of a series of three-residue sequences was investigated by substituting them into a well-characterized beta-hairpin peptide. The starting scaffold, bhpW, is a disulfide-cyclized 10-residue peptide that folds into a stable beta-hairpin with two antiparallel strands connected by a two-residue reverse turn. Substitution of the central two residues with the three-residue test sequences leads to less stable hairpins, as judged by thiol-disulfide equilibrium measurements. However, analysis of NMR parameters indicated that each molecule retains a significant folded population, and that the type of turn adopted by the three-residue sequence is the same in all cases. The solution structure of a selected peptide with a PDG turn contained an antiparallel beta-hairpin with a 3:5 type I + G1 bulge turn. Analysis of the energetic contributions of individual turn residues in the series of peptides indicates that substitution effects have significant context dependence, limiting the predictive power of individual amino acid propensities for turn formation. The most stable and least stable sequences were also substituted into a more stable disulfide-cyclized scaffold and a linear beta-hairpin scaffold. The relative stabilities remained the same, suggesting that experimental measurements in the bhpW context are a useful way to evaluate turn stability for use in protein design projects. Moreover, these scaffolds are capable of displaying a diverse set of turns, which can be exploited for the mimicry of protein loops or for generating libraries of reverse turns.     

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.     

Alternative type I and I' turn conformations in the beta8/beta9 beta-hairpin of human acidic fibroblast growth factor

Human acidic fibroblast growth factor (FGF-1) has a beta-trefoil structure, one of the fundamental protein superfolds. The X-ray crystal structures of wild-type and various mutant forms of FGF-1 have been solved in five different space groups: C2, C222(1), P2(1) (four molecules/asu), P2(1) (three molecules/asu), and P2(1)2(1)2(1). These structures reveal two characteristically different conformations for the beta8/beta9 beta-hairpin comprising residue positions 90-94. This region in the wild-type FGF-1 structure (P2(1), four molecules/asu), a his-tagged His93-->Gly mutant (P2(1), three molecules/asu) and a his-tagged Asn106-->Gly mutant (P2(1)2(1)2(1)) adopts a 3:5 beta-hairpin known as a type I (1-4) G1 beta-bulge (containing a type I turn). However, a his-tagged form of wild-type FGF-1 (C222(1)) and a his-tagged Leu44-->Phe mutant (C2) adopt a 3:3 beta-hairpin (containing a type I' turn) for this same region. A feature that distinguishes these two types of beta-hairpin structures is the number and location of side chain positions with eclipsed C(beta) and main-chain carbonyl oxygen groups (Psi is equivalent to +60 degrees). The effects of glycine mutations upon stability, at positions within the hairpin, have been used to identify the most likely structure in solution. Type I' turns in the structural data bank are quite rare, and a survey of these turns reveals that a large percentage exhibit crystal contacts within 3.0 A. This suggests that many of the type I' turns in X-ray structures may be adopted due to crystal packing effects.     

Solvent effects on the conformational transition of a model polyalanine peptide

We have investigated the folding of polyalanine by combining discontinuous molecular dynamics simulation with our newly developed off-lattice intermediate-resolution protein model. The thermodynamics of a system containing a single Ac-KA(14)K-NH(2) molecule has been explored by using the replica exchange simulation method to map out the conformational transitions as a function of temperature. We have also explored the influence of solvent type on the folding process by varying the relative strength of the side-chain's hydrophobic interactions and backbone hydrogen bonding interactions. The peptide in our simulations tends to mimic real polyalanine in that it can exist in three distinct structural states: alpha-helix, beta-structures (including beta-hairpin and beta-sheet-like structures), and random coil, depending upon the solvent conditions. At low values of the hydrophobic interaction strength between nonpolar side-chains, the polyalanine peptide undergoes a relatively sharp transition between an alpha-helical conformation at low temperatures and a random-coil conformation at high temperatures. As the hydrophobic interaction strength increases, this transition shifts to higher temperatures. Increasing the hydrophobic interaction strength even further induces a second transition to a beta-hairpin, resulting in an alpha-helical conformation at low temperatures, a beta-hairpin at intermediate temperatures, and a random coil at high temperatures. At very high values of the hydrophobic interaction strength, polyalanines become beta-hairpins and beta-sheet-like structures at low temperatures and random coils at high temperatures. This study of the folding of a single polyalanine-based peptide sets the stage for a study of polyalanine aggregation in a forthcoming paper.     

Energy landscape and dynamics of the beta-hairpin G peptide and its isomers: Topology and sequences

We have investigated free energy landscape [MM/PBSA + normal modes entropy] of permutations in the G peptide (41-56) from the protein G B1 domain by studying six isomers corresponding to moving the hydrophobic cluster along the beta-strands (toward the turn: T1, AGEWTYDDKTFTVTET; T2, GEDTWDYATFTVTKTE; T3, GEDDWTYATFTVTKTE; toward the end: E1, WTYDDAGETKTFTVT; E2, WEYTGDDATKTETFTV; E3, WTYEGDDATKTETFTV). The free energy terms include molecular mechanics energy, Poisson-Boltzmann electrostatic solvation energy, surface area solvation energy, and conformational entropy estimated by using normal mode analysis. From the wild type to T1, then T3, and finally T2, we see a progressively changing energy landscape, toward a less stable beta-hairpin structure. Moving the hydrophobic cluster outside toward the end region causes a greater change in the energy landscape. alpha-Helical instead of a beta-hairpin structure was the most stable form for the E2 isomer. However, no matter how much the sequence changes, for all variants studied, ideal "native" beta-hairpin topologies remain as minima (regardless of whether global or local) in the energy landscape. In general, we find that the energy landscape is dependent on the hydrophobic cluster topology and on the sequence. Our present study indicates that the key is the relative conformational energies of the different conformations. Changes in the sequence strongly modulate the relative stabilities of topologically similar regions in the energy landscape, rather than redefine the topology space. This finding is consistent with a population redistribution in the process of protein folding. The limited variation of topological space, compared with the number of possible sequence changes, may relate to the observation that the number of known protein folds are far less than the sequential allowance.     

Analysis of the factors that stabilize a designed two-stranded antiparallel beta-sheet

Autonomously folding beta-hairpins (two-strand antiparallel beta-sheets) have become increasingly valuable tools for probing the forces that control peptide and protein conformational preferences. We examine the effects of variations in sequence and solvent on the stability of a previously designed 12-residue peptide (1). This peptide adopts a beta-hairpin conformation containing a two-residue loop (D-Pro-Gly) and a four-residue interstrand sidechain cluster that is observed in the natural protein GB1. We show that the conformational propensity of the loop segment plays an important role in beta-hairpin stability by comparing 1 with (D)P--> N mutant 2. In addition, we show that the sidechain cluster contributes both to conformational stability and to folding cooperativity by comparing 1 with mutant 3, in which two of the four cluster residues have been changed to serine. Thermodynamic analysis suggests that the high loop-forming propensity of the (D)PG segment decreases the entropic cost of beta-hairpin formation relative to the more flexible NG segment, but that the conformational rigidity of (D)PG may prevent optimal contacts between the sidechains of the GB1-derived cluster. The enthalpic favorability of folding in these designed beta-hairpins suggests that they are excellent scaffolds for studying the fundamental mechanisms by which amino acid sidechains interact with one another in folded proteins.     

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.     

Polymorphism of β2-microglobulin amyloid fibrils manifested by ultrasonication-enhanced fibril formation in trifluoroethanol

The polymorphic property of amyloid structures has been focused on as a molecular basis of the presence and propagation of different phenotypes of amyloid diseases, although little is known about the molecular mechanism for expressing diverse structures from only one protein sequence. Here, we have found that, in combination with an enhancing effect of ultrasonication on nucleation, β(2)-microglobulin, a protein responsible for dialysis-related amyloidosis, generates distinct fibril conformations in a concentration-dependent manner in the presence of 2,2,2-trifluoroethanol (TFE). Although the newly formed fibrils all exhibited a similar needle-like morphology with an extensive cross-β core, as suggested by Fourier transform infrared absorption spectra, they differed in thioflavin T intensity, extension kinetics, and tryptophan fluorescence spectra even in the same solvents, representing polymorphic structures. The hydrophobic residues seemed to be more exposed in the fibrils originating at higher concentrations of TFE, as indicated by the increased binding of 1-anilinonaphthalene-8-sulfonic acid, suggesting that the modulation of hydrophobic interactions is critical to the production of polymorphic amyloid structures. Interestingly, the fibrils formed at higher TFE concentrations showed significantly higher stability against guanidium hydrochloride, the perturbation of ionic strength, and, furthermore, pressurization. The cross-β structure inside the fibrils seems to have been more idealized, resulting in increased stability when nucleation occurred in the presence of the alcohol, indicating that a weaker contribution of hydrophobic interactions is intrinsically more amenable to the formation of a non-defective amyloid structure.     

Competing intrachain interactions regulate the formation of beta-sheet fibrils in bovine PrP peptides

At the heart of the pathogenesis of transmissible spongiform encephalopathies (TSEs), such as BSE, scrapie, and Creutzfeldt-Jakob disease, lies a poorly understood structural rearrangement of PrP, an abundant glycoprotein of the nervous and lymphoid systems. The normal form (PrP(C)), rich in alpha-helix, converts into an aberrant beta-sheet-dominated form (PrP(Sc)), which seems to be at the center of the pathotoxic symptoms observed in TSEs. To understand this process better at a molecular level, we have studied the interactions between different peptides derived from bovine PrP and their structural significance. We show that two unstructured peptides derived from the central region of bovine PrP, residues 115-133 and 140-152, respectively, interact stoichiometrically under physiological conditions to generate beta-sheet-dominated fibrils. However, when both peptides are incubated in the presence of a third peptide derived from an adjoining alpha-helical region (residues 153-169), the formation of beta-sheet-rich fibrils is abolished. These data indicate that native PrP(C) helix 1 might inhibit the strong intrinsic beta-sheet-forming propensity of sequences immediately N-terminal to the globular core of PrP(C), by keeping in place intrachain interactions that would prevent these amyloidogenic regions from triggering aggregation. Moreover, these results indicate new ways in which PrP(Sc) formation could be prevented.     

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.     

Molecular Mechanism of Thioflavin-T Binding to the Surface of β-Rich Peptide Self-Assemblies

A number of small organic molecules have been developed that bind to amyloid fibrils, a subset of which also inhibit fibrillization. Among these, the benzothiol dye Thioflavin-T (ThT) has been used for decades in the diagnosis of protein-misfolding diseases and in kinetic studies of self-assembly (fibrillization). Despite its importance, efforts to characterize the ThT-binding mechanism at the atomic level have been hampered by the inherent insolubility and heterogeneity of peptide self-assemblies. To overcome these challenges, we have developed a minimalist approach to designing a ThT-binding site in a "peptide self-assembly mimic” (PSAM) scaffold. PSAMs are engineered water-soluble proteins that mimic a segment of β-rich peptide self-assembly, and they are amenable to standard biophysical techniques and systematic mutagenesis. The PSAM β-sheet contains rows of repetitive amino acid patterns running perpendicular to the strands (cross-strand ladders) that represent a ubiquitous structural feature of fibril-like surfaces. We successfully designed a ThT-binding site that recapitulates the hallmarks of ThT-fibril interactions by constructing a cross-strand ladder consisting of contiguous tyrosines. The X-ray crystal structures suggest that ThT interacts with the β-sheet by docking onto surfaces formed by a single tyrosine ladder, rather than in the space between adjacent ladders. Systematic mutagenesis further demonstrated that tyrosine surfaces across four or more β-strands formed the minimal binding site for ThT. Our work thus provides structural insights into how this widely used dye recognizes a prominent subset of peptide self-assemblies, and proposes a strategy to elucidate the mechanisms of fibril-ligand interactions.