Folding of the 25 residue Abeta(12-36) peptide in TFE/water: temperature-dependent transition from a funneled free-energy landscape to a rugged one

The free-energy landscape of the Alzheimer beta-amyloid peptide Abeta(12-36) in a 40% (v/v) 2,2,2-trifluoroethanol (TFE)/water solution was determined by using multicanonical molecular dynamics simulations. Simulations using this enhanced conformational sampling technique were initiated from a random unfolded polypeptide conformation. Our simulations reliably folded the peptide to the experimental NMR structure, which consists of two linked helices. The shape of the free energy landscape for folding was found to be strongly dependent on temperature: Above 325 K, the overall shape was funnel-like, with the bottom of the funnel coinciding exactly with the NMR structure. Below 325 K, on the other hand, the landscape became increasingly rugged, with the emergence of new conformational clusters connected by low free-energy pathways. Finally, our simulations reveal that water and TFE solvate the polypeptide in different ways: The hydrogen bond formation between TFE and Abeta was enhanced with decreasing temperature, while that between water and Abeta was depressed.     

Stability of cyclic beta-hairpins: asymmetric contributions from side chains of a hydrogen-bonded cross-strand residue pair

Amino acid structural propensities measured in "host-guest" model studies are often used in protein structure prediction or to choose appropriate residues in de novo protein design. While this concept has proven useful for helical structures, it is more difficult to apply successfully to beta-sheets. We have developed a cyclic beta-hairpin scaffold as a host for measurement of individual residue contributions to hairpin structural stability. Previously, we have characterized substitutions in non-backbone-hydrogen-bonded strand sites; relative stability differences measured in the cyclic host are highly predictive of changes in folding free energy for linear beta-hairpin peptides. Here, we examine the hydrogen-bonded strand positions of our host. Surprisingly, we find a large favorable contribution to stability from a valine (or isoleucine) substitution immediately preceding the C-terminal cysteine of the host peptide, but not at the cross-strand position of the host or in either strand of a folded linear beta-hairpin (trpzip peptide). Further substitutions in the peptides and NMR structural analysis indicate that the stabilizing effect of valine is general for CX(8)C cyclic hairpins and cannot be explained by particular side-chain-side-chain interactions. Instead, a localized decrease in twist of the peptide backbone on the N-terminal side of the cysteine allows the valine side chain to adopt a unique conformation that decreases the solvent accessibility of the peptide backbone. The conformation differs from the highly twisted (coiled) conformation of the trpzip hairpins and is more typical of conformations present in multistranded beta-sheets. This unexpected structural fine-tuning may explain why cyclic hairpins selected from phage-displayed libraries often have valine in the same position, preceding the C-terminal cysteine. It also emphasizes the diversity of structures accessible to beta-strands and the importance of considering not only "beta-propensity", but also hydrogen-bonding pattern and strand twist, when designing beta structures. Finally, we observe correlated, cooperative stabilization from side-chain substitutions on opposite faces of the hairpin. This suggests that cooperative folding in beta-hairpins and other small beta-structures is driven by cooperative strand-strand association.     

Beta-hairpin folding by a model amyloid peptide in solution and at an interface

The development of specific agents against amyloidoses requires an understanding of the conformational distribution of fibrillogenic peptides at a microscopic level. Here, I present molecular dynamics simulations of the model amyloid peptide LSFD with sequence LSFDNSGAITIG-NH2 in explicit water and at a water/vapor interface for a total time scale of approximately 1.8 micros. An extended structure was used as initial peptide configuration. At approximately 290 K, solvated LSFD was kinetically trapped in diverse misfolded beta-sheet/coil conformations. At 350 K, in contrast, the same type II' beta-hairpin in equilibrium with less ordered but also U-shaped conformations was observed for the core residues DNSGAITI in solution and at the interface in multiple independent simulations. The most stable structural unit of the beta-hairpin was the two residue turn (GA). The core residues exhibited a well-defined folded state in which the beta-hairpin was stabilized by a hydrogen bond between the side chain of Asn-385 and the main chain carbonyl group of Gly-387. My results suggest that beta-sheet conformations indicated from previous Fourier-transform infrared spectroscopy measurements immediately after preparation of the peptide solution may not arise from protofilaments as speculated by others but are a property of LSFD monomers. In addition, combined with previous results from X-ray scattering, my findings suggest that interfacial aggregation of LSFD implies a transition from U-shaped to extended peptide conformations. This work including the first simulations of reversible beta-hairpin folding at an interface is an essential step toward a microscopic understanding of interfacial peptide folding and self-assembly. Knowledge of the main conformation of the peptide core may facilitate the design of possible inhibitors of LSFD aggregation as a test ground for future computational therapeutic strategies against amyloid diseases.     

Absence of reptation in the high-temperature folding of the trpzip2 beta-hairpin peptide

We have carried out extensive all atom explicit solvent simulations of the high-temperature folding and unfolding of the trpzip2 beta-hairpin peptide and examined the resulting trajectories for evidence of folding via a reptation mechanism. Over 300 microcanonical simulations of 10 ns each were initiated from a Boltzmann ensemble of conformations at 425 K. Though we observed numerous folding and unfolding events, no evidence of reptation was found. The diffusional dynamics of the peptide are orders of magnitude faster than any observed reptation-like motion. Our data suggest that the dominant mechanisms for beta-hairpin folding under these conditions are hydrophobic collapse and turn formation, and that rearrangements occur via significant expansion of the polypeptide chain.     

Helix formation in alpha,gamma- and beta,gamma-hybrid peptides: theoretical insights into mimicry of alpha- and beta-peptides

Alpha,gamma- and beta,gamma-hybrid peptides, which are composed of two different homologous amino acid constituents in alternate order, are suggested as novel classes of peptide foldamers. On the basis of a systematic conformational search employing the methods of ab initio MO theory, the possibilities for the formation of periodic secondary structures in these systems are described. The conformational analysis provides a great number of helix conformers widely differing in energy, which can be arranged into three groups: (i) helices with all hydrogen bonds formed in forward direction along the sequence, (ii) helices with all hydrogen bonds in backward direction, and (iii) helices with alternate hydrogen-bond directions (mixed or beta-helices). Most stable are representatives of beta-helices, but their stability decreases considerably in more polar environments in comparison to helix conformers from the other two classes. There is a great similarity between the overall topology of the most stable hybrid peptide helices and typical helices of peptides which are exclusively composed of a single type of homologous amino acids. Thus, the helices of the beta,gamma-hybrid peptides mimic perfectly those of the native alpha-peptides as, for instance, the well-known alpha-helix, whereas the most stable helix conformers of alpha,gamma-hybrid peptides correspond well to the overall structure of beta-peptide helices. The two suggested novel hybrid peptide classes expand considerably the pool of peptide foldamers and may be promising tools in peptide design and in material sciences.     

Peptide pinwheels

Electrospraying a mixture of Ac-(GA)7K and Ac-A(GA)7K (Ac = acetyl, G = glycine, A = alanine, and K = lysine) peptides produces strong signals for unsolvated dimers and trimers. The conformations of these multimers have been examined with use of ion mobility measurements in conjunction with molecular dynamics simulations. The results suggest that the trimers adopt a pinwheel arrangement of helices with the C-termini tethered together by the protonated lysine side chain from one peptide interacting with the C-terminus of a neighboring helix. This arrangement leads to a cooperative electrostatic stabilization of all the helices through the interaction of the combined charge with the helix dipoles. The dimer adopts a related V-shaped arrangement of helices which is also cooperatively stabilized.     

Helix formation in the polymer brush

This work considers the physics of a brush formed by polymers capable of undergoing a helix-coil transition. A self-consistent field approximation for strongly stretched polymer chains is used in combination with a lattice model of the interaction energy in helix-coil mixtures. Crowding-induced chain stretching stabilizes helix formation at moderate tethering densities while high tethering density causes sufficiently strong stretching to unravel segments of the helix, resulting in distinct layers of monomer density and helical content. Compared to a random-coil brush at low-to-moderate tethering density, a helicogenic brush is less resistant to compression in the direction perpendicular to stretching due to easy alignment of helices and fewer unfavorable interactions between helical segments. At higher tethering density, the abovementioned stretch-induced decrease in helical content resists further compression. The proposed model is useful for understanding an emerging class of biomaterials that utilize helix-forming polymer brushes to induce shape changes or to stabilize biofunctional helical peptide sequences.     

Glycopeptides related to beta-endorphin adopt helical amphipathic conformations in the presence of lipid bilayers

A series of glycosylated endorphin analogues designed to penetrate the blood-brain barrier (BBB) have been studied by circular dichroism and by 2D-NMR in the presence of water; TFE/water; SDS micelles; and in the presence of both neutral and anionic bicelles. In water, the glycopeptides showed only nascent helix behavior and random coil conformations. Chemical shift indices and nuclear Overhauser effects (NOE) confirmed helices in the presence of membrane mimics. NOE volumes provided distance constraints for molecular dynamics calculations used to provide detailed backbone conformations. In all cases, the glycopeptides were largely helical in the presence of membrane bilayer models (micelles or bicelles). Plasmon waveguide resonance (PWR) studies showed hen egg phosphatidyl choline (PC) bilayers produce amphipathic helices laying parallel to the membrane surface, with dissociation constants (K(D)) in the low nanomolar to micromolar concentration range. Two low-energy states are suggested for the glycosylated endorphin analogues, a flexible aqueous state and a restricted membrane bound state. Strong interactions between the glycopeptide amphipaths and membranes are crucial for penetration of the BBB via an endocytotic mechanism (transcytosis).     

The strength with which a peptide group can form a hydrogen bond varies with the internal conformation of the polypeptide chain

The strength of the H-bond formed between a dipeptide and a proton acceptor molecule is assessed by correlated ab initio quantum calculations for a broad range of different conformations of the dipeptide. The H-bond energy is very sensitive to the internal (phi,psi) angles, even when the geometry of the H-bond does not vary significantly from one conformation to another. This result indicates that the peptide NH is a much less potent proton donor in certain conformations than in others. In particular, extended conformations of a polypeptide are capable of only weak H-bonds. Thus, the interstrand NH...O H-bonds in parallel and antiparallel beta-sheets are expected to be significantly weaker than those found in other conformations, such as helices, ribbons, and beta-bends, even if the H-bond geometries are similar.     

Molecular conformation and packing of peptide beta hairpins in the solid state: structures of two synthetic octapeptides containing 1-aminocycloalkane-1-carboxylic acid residues at the i+2 position of the beta turn

Peptide beta-hairpin formation is facilitated by centrally positioned D-Pro-Xxx segments. The synthetic peptides Boc-Leu-Phe-Val-D-Pro-Ac(6)c-Leu-Phe-Val-OMe (1) and Boc-Leu-Phe-Val-D-Pro-Ac(8)c-Leu-Phe-Val-OMe (2) were synthesized in order to explore the role of bulky 1-aminocycloalkane-1-carboxylic acid residues (Ac(n)c, where n is the number of carbon atoms in the ring), at the i+2 position of the nucleating beta turn in peptide beta hairpins. Peptides 1 and 2 crystallize in the monoclinic space group P2(1) with two molecules in the asymmetric unit. The crystal structures of 1 and 2 provide conformational parameters for four peptide hairpin molecules. In all cases, the central segments adopts a type II' beta-turn conformation, and three of the four possible cross-strand hydrogen bonds are observed. Fraying of the hairpins at the termini is accompanied by the observation of NHpi interaction between the Leu(1)NH group and Phe(7) aromatic group. Cross strand stabilizing interactions between the facing residues Phe(2) and Phe(7) are suggested by the observed orientation of aromatic rings. Anomalous far-UV CD spectra observed in solution suggest that close proximity of the Phe rings is maintained even in isolated molecules. In both peptides 1 and 2, the asymmetric unit consists of approximately orthogonal hairpins, precluding the formation of a planar beta-sheet arrangement in the solid state. Solvent molecules, one dioxane and one water in 1, three water molecules in 2, mediate peptide association. A comparison of molecular conformation and packing motifs in available beta-hairpin structures permits delineation of common features. The crystal structures of beta-hairpin peptides provide a means of visualizing different modes of beta-sheet packing, which may be relevant in developing models for aggregates of polypeptides implicated in disease situations.     

High-pressure effect on the dynamics of solvated peptides

The dynamics of peptides has a direct connection to how quickly proteins can alter their conformations. The speed of exploring the free energy landscape depend on many factors, including the physical parameters of the environment, such as pressure and temperature. We performed a series of molecular dynamics simulations to investigate the pressure-temperature effects on peptide dynamics, especially on the torsional angle and peptide-water hydrogen bonding (H-bonding) dynamics. Here, we show that the dynamics of the omega angle and the H-bonding dynamics between water and the peptide are affected by pressure. At high temperature (500 K), both the dynamics of the torsional angle ω and H-bonding slow down significantly with increasing pressure, interestingly, at approximately the same rate. However, at a lower temperature of 300 K, the observed trend on H-bonding dynamics as a function of pressure reverses, i.e., higher pressure speeds up H-bonding dynamics.     

Structural effects on the formation of proton and alkali metal ion adducts of apolar,neutral peptides: electrospray ionization mass spectrometry and Ab initio theoretical studies

Apolar, neutral peptides have been shown to ionize extremely well under the conditions used for electrospray ionization mass spectrometry (ESIMS). Peptides for which the conformations have been independently determined in solution and in crystals have been examined by ESIMS. Studies of peptide helices ranging from 7 to 18 residues reveal that shorter helices yield exclusively singly charged ions, while in larger helices multiply charged species are detectable. Multiple sites for protonation/metallation are introduced in the helix by proline insertion or by changing the chirality in the residue. The preferred site of cation binding to helices may be the C-terminus end, where three free CO groups are available for chelation. Ab initio and DFT calculations at several levels have been carried out for the binding of H+, Li+, Na+, and K+ to CHO-(Gly)3)-OMe. The results reveal that metallation in helices is favoured by chelation to carbonyl groups at the C-terminus, while protonation involved two carbonyl groups and thus favour a 10-membered cyclic hydrogen-bonded structure. In -strands, metallation/protonation occurs at isolated carbonyl groups. Collision induced fragmentation of hydrophobic peptides under ESI conditions reveals that helix fragmentation occurs predominantly from the C-terminus, while in -hairpins cleavage occurs simultaneously at multiple sites.     

The influence of different treatments of electrostatic interactions on the thermodynamics of folding of peptides

Replica exchange molecular dynamics simulations were performed to investigate the effects of different electrostatic treatments on the structure and thermodynamics of a small beta-hairpin forming peptide. Three different electrostatic schemes were considered: regular cutoffs, generalized reaction field (GRF), and particle mesh Ewald (PME), with the peptide modeled using OPLS/AA all-atom force field with explicit TIP3P water. Both the GRF and PME methods yielded results consistent with experiment, with free energy surfaces displaying a single minimum corresponding to the native beta-hairpin structure. In contrast, use of straight cutoffs led to the population of an additional local minimum corresponding to nonhairpin conformations that compete with the formation of the native beta-hairpin at low temperatures. This extra minimum would not be apparent in conventional constant-temperature molecular dynamics simulations run for a few nanoseconds. This result points to the critical need of careful sampling of conformational space to assess the quality of different numerical treatments of long-range forces. While differences emerged in the nature of the unfolded states populated using PME and GRF approaches, simulations on the beta-hairpin forming peptide and on two additional control peptides indicate that the GRF treatment of electrostatics offers a satisfactory compromise between accuracy and computational speed for the identification of low-energy conformations. A GRF-based approach emerges as a viable means for treating larger biological systems that would be prohibitively costly to simulate using PME methods.     

Probing the formation of stable tertiary structure in a model miniprotein at atomic resolution: determinants of stability of a helical hairpin

The minimal model system to study the basic principles of protein folding is the hairpin. The formation of beta-hairpins, which are the basic components of antiparallel beta-sheets, has been studied extensively in the past decade, but much less is known about helical hairpins. Here, we probe hairpin formation between a polyproline type-II helix and an alpha-helix as present in the natural miniprotein peptide YY (PYY). Both turn sequence and interactions of aromatic side chains from the C-terminal alpha-helix with the pockets formed by N-terminal Pro residues are shown by site-directed mutagenesis and solution NMR spectroscopy in different solvent systems to be important determinants of backbone dynamics and hairpin stability, suggesting a close analogy with some beta-hairpin structures. It is shown that multiple relatively weak contacts between the helices are necessary for the formation of the helical hairpin studied here, whereas the type-I beta-turn acts like a hinge, which through certain single amino acid substitutions is destabilized such that hairpin formation is completely abolished. Denaturation and renaturation of tertiary structure by temperature or cosolvents were probed by measuring changes of chemical shifts. Folding of PYY is both reversible and cooperative as inferred from the sigmoidal denaturation curves displayed by residues at the interface of the helical hairpin. Such miniproteins thus feature an important hallmark of globular proteins and should provide a convenient system to study basic aspects of helical hairpin folding that are complementary to those derived from studies of beta-hairpins.     

Water molecule adsorption on short alanine peptides: how short is the shortest gas-phase alanine-based helix?

Water adsorption measurements have been performed under equilibrium conditions for unsolvated Ac-A(n)K+H(+) and Ac-KA(n)+H(+) peptides with n = 4 - 10. Previous work on larger alanine peptides has shown that two dominant conformations (helices and globules) are present for these peptides and that water adsorbs much more strongly to the globules than to the helices. All the Ac-KA(n)+H(+) peptides studied here (which are expected to be globular) adsorb water strongly, and so do the Ac-A(n)K+H(+) peptides with n < 8. However, for Ac-A(n)K+H(+) with n = 8-10 there is a substantial drop in the propensity to adsorb water. This result suggests that Ac-A(8)K+H(+) is the smallest Ac-A(n)K+H(+) peptide to have a significant helical content in the gas phase. Water adsorption measurements for Ac-V(n)K+H(+) and Ac-L(n)K+H(+) with n = 5-10 suggest that the helix emerges at n = 8 for these peptides as well.     

A free-energy landscape for coupled folding and binding of an intrinsically disordered protein in explicit solvent from detailed all-atom computations

The N-terminal repressor domain of neural restrictive silencer factor (NRSF) is an intrinsically disordered protein (IDP) that binds to the paired amphipathic helix (PAH) domain of mSin3. An NMR experiment revealed that the minimal binding unit of NRSF is a 15-residue segment that adopts a helical structure upon binding to a cleft of mSin3. We computed a free-energy landscape of this system by an enhanced conformational sampling method, all-atom multicanonical molecular dynamics. The simulation started from a configuration where the NRSF segment was fully disordered and distant from mSin3 in explicit solvent. In the absence of mSin3, the disordered NRSF segment thermally fluctuated between hairpins, helices, and bent structures. In the presence of mSin3, the segment bound to mSin3 by adopting the structures involved in the isolated state, and non-native and native complexes were formed. The free-energy landscape comprised three superclusters, and free-energy barriers separated the superclusters. The native complex was located at the center of the lowest free-energy cluster. When NRSF landed in the largest supercluster, the generated non-native complex moved on the landscape to fold into the native complex, by increasing the interfacial hydrophobic contacts and the helix content. When NRSF landed in other superclusters, the non-native complex overcame the free-energy barriers between the various segment orientations in the binding cleft of mSin3. Both population-shift and induced-fit (or induced-folding) mechanisms work cooperatively in the coupled folding and binding. The diverse structural adaptability of NRSF may be related to the hub properties of the IDP.     

UV resonance Raman measurements of poly-L-lysine's conformational energy landscapes: dependence on perchlorate concentration and temperature

UV resonance Raman spectroscopy has been used to determine the conformational energy landscape of poly-L-lysine (PLL) in the presence of NaClO4 as a function of temperature. At 1 degree C, in the presence of 0.83 M NaClO4, PLL shows an approximately 86% alpha-helix-like content, which contains alpha-helix and pi-bulge/helix conformations. The high alpha-helix-like content of PLL occurs because of charge screening due to strong ion-pair formation between ClO4- and the lysine side chain -NH3+. As the temperature increases from 1 to 60 degrees C, the alpha-helix and pi-bulge/helix conformations melt into extended conformations (PPII and 2.51-helix). We calculate the Psi Ramachandran angle distribution of the PLL peptide bonds from the UV Raman spectra which allows us to calculate the PLL (un)folding energy landscapes along the Psi reaction coordinate. We observe a basin in the Psi angle conformational space associated with alpha-helix and pi-bulge/helix conformations and another basin for the extended PPII and 2.51-helical conformations.     

U-shaped caveolin-1 conformations are tightly regulated by hydrogen bonds with lipids

The structure and dynamics of a truncated (residues 82–136) caveolin-1 (Cav1) construct having a helix-break-helix motif are explored by both all-atom free energy and molecular dynamics (MD) simulations in an explicit bilayer membrane. Two stable Cav1 conformations with small (LB-Cav1) and large hinge angles (RB-Cav1) between two helices are identified although their relative free energy cannot be reliably estimated due to the sampling issues. RB-Cav1s contain one or two lipids residing between the helices that are hydrogen bonded (h-bonded) to both helices in a multidentate fashion. LB-Cav1s show the helices with mono-dentate lipid h-bond interactions or multidentate interactions limited to a single helix at most. The two conformational states of Cav1 remain their initial state during 2-μs MD simulation, suggesting that there is a significant hidden barrier (other than the insertion depth of Cav1 and its hinge angle) and the Cav1 conformational states are tightly regulated by the h-bonds between Cav1 and lipids along with the associated lipid rearrangement during the course of Cav1 conformational changes. © 2019 Wiley Periodicals, Inc.     

Helix-turn-helix motifs in unsolvated peptides

The conformations of unsolvated Ac-A14KG3A14K + 2H+ (Ac = acetyl, A = alanine, K = lysine, G = glycine) have been examined by ion mobility measurements and molecular dynamics simulations. This peptide was designed as a model helix-turn-helix motif. It was found to adopt three distinct geometries which were assigned to an extended helical conformation which is only stable at low temperatures (<230 K), a relatively high energy but metastable structure with exchanged lysines, and a coiled-coil. The coiled coil (which consists of an antiparallel arrangement of two helical alanine sections linked by a flexible glycine loop) is the dominant conformation. For temperatures >350 K, the experimental results indicate the helices uncouple and the loop randomizes. From equilibrium constants determined for this helix coupling right arrow over left arrow uncoupling transition, we found DeltaH degrees = -45 kJ mol-1 and DeltaS degrees = 114 J K-1 mol-1. -DeltaH degrees is essentially the enthalpy change for docking the two helices together while DeltaS degrees is essentially the entropy change for freeing up the glycine loop.