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Streptococcus mutans is a bacterial species that predominates in the oral microbiome. S. mutans binds to the tooth surface, metabolizes sugars and produces acid, leading to cavity formation. S. mutans can also cause infectious endocarditis. Recent evidence suggests that S. mutans biofilms contain amyloid fibrils. Amyloids are insoluble fibrillar protein aggregates, and bacteria use functional amyloids to improve robustness of their biofilms. While the functional amyloids in bacteria such as Escherichia coli and Staphylococcus aureus have been heavily investigated, little is known about the mechanism of S. mutans amyloid formation. Previous results from our laboratory with the amyloidogenic proteins and peptides from the aforementioned bacteria and other mammalian amyloid systems suggest that amyloid formation progresses via an intermediate that adopts a unique secondary structure-α-sheet. De novo designed peptides with alternating l- and d-amino acid also adopt an α-sheet secondary structure and inhibit amyloid formation by binding to soluble oligomeric species during amyloidogenesis. Inhibition of fibrillization by α-sheet peptides suggests the presence of α-sheet during amyloid formation. To investigate the mechanism of functional amyloid formation in S. mutans, α-sheet peptides were compared to epigallocatechin gallate for their ability to inhibit fibril formation in S. mutans. Inhibition was demonstrated in a biofilm plate assay and on hydroxyapatite surfaces both in S. mutans alone and in bacteria from human saliva. The observed inhibition suggests that an α-sheet mediated mechanism may be operative during functional amyloid formation., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

There has been much interest in synthetic peptides as inhibitors of aggregation associated with amyloid diseases. Of particular interest are compounds that target the cytotoxic soluble oligomers preceding the formation of mature, nontoxic fibrils. This study explores physical and chemical differences between two de novo-designed peptides that share an identical primary structure but differ in backbone chirality at six key positions. We show that the presence of alternating l/d-amino acid motifs dramatically increases aqueous solubility, enforces α-sheet secondary structure, and inhibits aggregation of the β-amyloid peptide implicated in Alzheimer's disease, in addition to neutralizing its cytotoxicity. In contrast, the all-l-amino acid isomer does not form α-sheet structure and is insoluble and inactive.

Previous studies suggest that the toxic soluble-oligomeric form of different amyloid proteins share a common backbone conformation, but the amorphous nature of this oligomer prevents its structural characterization by experiment. Based on molecular dynamics simulations we proposed that toxic intermediates of different amyloid proteins adopt a common, nonstandard secondary structure, called α-sheet. Here we report the experimental characterization of peptides designed to be complementary to the α-sheet conformation observed in the simulations. We demonstrate inhibition of aggregation in two different amyloid systems, β-amyloid peptide (Aβ) and transthyretin, by these designed α-sheet peptides. When immobilized the α-sheet designs preferentially bind species from solutions enriched in the toxic conformer compared with non-aggregated, nontoxic species or mature fibrils. The designs display characteristic spectroscopic signatures distinguishing them from conventional secondary structures, supporting α-sheet as a structure involved in the toxic oligomer stage of amyloid formation and paving the way for novel therapeutics and diagnostics.DOI: http://dx.doi.org/10.7554/eLife.01681.001.

It has been suggested that denatured proteins are predisposed toward the left-handed polyproline II (P(II)) conformation. One possible source of P(II) stability in the denatured state is water bridges. Water bridges are networks of water molecules that link nearby hydrogen bond acceptors and/or donors on proteins. On the basis of the proposed behavior of P(II) and water bridges, the propensity of a residue to participate in water bridges should be correlated with its P(II) propensity. To test this hypothesis, we analyzed the following data sets: 2351 high-resolution crystal structures, and the native and denatured states of 188 different proteins from all-atom, explicit-solvent molecular dynamics (MD) simulations, which are part of our Dynameomics effort. We found that water bridges do not explain the high frequency of P(II) in denatured states; such bridges are less frequent around P(II) than around other conformations. Thus, this analysis casts doubt on water bridges as a dominant factor determining the residue-based P(II) propensities.

Rarely is any solution simply solute and water. In vivo, solutes, such as proteins and nucleic acids, swim in a sea of water, salts, ions, small molecules, and lipids, not to mention other macromolecules. In vitro, virtually all solutions contain a mixture of aqueous solvents, or "cosolvents" [i.e., solvent(s) in addition to water], that can alter the dynamics, behavior, solubility, and stability of proteins and nucleic acids. We have developed models for a number of cosolvents, including the denaturant urea and the small chemical chaperone trimethylamine N-oxide (TMAO). This chapter examines the models for these two cosolvents in the context of experimental data. The direct and indirect effects of these molecules on water and protein are studied with molecular dynamics simulations. These observations and conclusions are drawn from simulations of these molecules in pure water and as a cosolvent for the protein chymotrypsin inhibitor 2. Urea-induced denaturation occurs initially through attack of the protein by water and hydration of hydrophobic protein moieties as a result of disruption of the hydrogen bonding network of water by urea. This indirect denaturing effect of urea is followed by more direct action as urea replaces some waters involved in the initial hydration of the hydrophobic core and subsequently binds to polar residues and the protein main chain to compete with the intraprotein hydrogen bonds. In the case of TMAO, we find that it encourages water-water interactions, thereby stabilizing the protein as a result of the increased penalty for the hydration of hydrophobic residues.

Several neurodegenerative diseases are linked to expanded repeats of glutamine residues, which lead to the formation of amyloid fibrils and neuronal death. The length of the repeats correlates with the onset of Huntington's disease, such that healthy individuals have <38 residues and individuals with >38 repeats exhibit symptoms. Because it is difficult to obtain atomic-resolution structural information for poly(l-glutamine) (polyQ) in aqueous solution experimentally, we performed molecular dynamics simulations to investigate the conformational behavior of this homopolymer. In simulations of 20-, 40-, and 80-mer polyQ, we observed the formation of the "alpha-extended chain" conformation, which is characterized by alternating residues in the alpha(L) and alpha(R) conformations to yield a sheet. The structural transition from disordered random-coil conformations to the alpha-extended chain conformation exhibits modest length and temperature dependence, in agreement with the experimental observation that aggregation depends on length and temperature. We propose that fibril formation in polyQ may occur through an alpha-sheet structure, which was proposed by Pauling and Corey. Also, we propose an atomic-resolution model of how the inhibitory peptide QBP1 (polyQ-binding peptide 1) may bind to polyQ in an alpha-extended chain conformation to inhibit fibril formation.

Molecular dynamics (MD) simulations are the classic single-molecule "experiments," providing atomic-resolution structural and dynamic information. However, the single-molecule nature of the technique has also been its shortcoming, with frequent criticisms of sampling inadequacies and questions regarding the ensemble behavior of large numbers of molecules. Given the increase in computer power, we now address this issue by performing a large number of simulations and comparing individual and ensemble properties. One hundred independent MD simulations of the protein chymotrypsin inhibitor 2 were carried out for 20 ns each at 498 K in water to more fully describe the potentially diverse routes of protein unfolding and investigate how representative a single trajectory can be. Rapid unfolding was observed in all cases with the trajectories distributed about an average "ensemble" path in which secondary and tertiary structure was lost concomitantly, with tertiary structure loss occurring slightly faster. Individual trajectories did, however, sample conformations far from the average path with very heterogeneous time-dependent properties. Nevertheless, all of the simulations but one followed the average ensemble pathway, such that a small number of simulations (5-10) are sufficient to capture the average properties of these states and the unfolding pathway.

To better characterize the transition state for folding/unfolding and its sensitivity to environmental changes, we have run multiple molecular dynamics simulations of chymotrypsin inhibitor 2 (CI2) under varying solvent conditions and temperature. The transition state structures agree well with experiment, and are similar under all of the conditions investigated here. Increasing the temperature leads to some movement in the position of the transition state along several reaction coordinates, as measured by changes in properties of the transition state structures. These structural changes are in the direction of a more native-like transition state as denaturation conditions become more severe, as expected for a Hammond effect. These structural changes are not, however, reflected in the global structure as measured by the total number of contacts or the average S-values. These results suggest that the small changes in average Phi-values with temperature seen by experiment may be due to an increase in the sensitivity of the transition state to mutation rather than a change in the average structure of the transition state. A simple analysis of the rates of unfolding indicates that the free energy barrier to unfolding decreases with increasing temperature, but even in our very high temperature simulations there is a small free energy barrier.

The correct treatment of van der Waals and electrostatic nonbonded interactions in molecular force fields is essential for performing realistic molecular dynamics (MD) simulations of solvated polypeptides. The most computationally tractable treatment of nonbonded interactions in MD utilizes a spherical distance cutoff (typically, 8-12 A) to reduce the number of pairwise interactions. In this work, we assess three spherical atom-based cutoff approaches for use with all-atom explicit solvent MD: abrupt truncation, a CHARMM-style electrostatic shift truncation, and our own force-shifted truncation. The chosen system for this study is an end-capped 17-residue alanine-based alpha-helical peptide, selected because of its use in previous computational and experimental studies. We compare the time-averaged helical content calculated from these MD trajectories with experiment. We also examine the effect of varying the cutoff treatment and distance on energy conservation. We find that the abrupt truncation approach is pathological in its inability to conserve energy. The CHARMM-style shift truncation performs quite well but suffers from energetic instability. On the other hand, the force-shifted spherical cutoff method conserves energy, correctly predicts the experimental helical content, and shows convergence in simulation statistics as the cutoff is increased. This work demonstrates that by using proper and rigorous techniques, it is possible to correctly model polypeptide dynamics in solution with a spherical cutoff. The inherent computational advantage of spherical cutoffs over Ewald summation (and related) techniques is essential in accessing longer MD time scales.

The conformational equilibrium between 3(10)- and alpha-helical structure has been studied via high-resolution NMR spectroscopy by Millhauser and coworkers using the MW peptide Ac-AMAAKAWAAKA AAARA-NH2. Their 750-MHz nuclear Overhauser effect spectroscopy (NOESY) spectra were interpreted to reflect appreciable populations of 3(10)-helix throughout the peptide, with the greatest contribution at the N and C termini. The presence of simultaneous alphaN(i,i + 2) and alphaN(i,i + 4) NOE cross-peaks was proposed to represent conformational averaging between 3(10)- and alpha-helical structures. In this study, we describe 25-nsec molecular dynamics simulations of the MW peptide at 298 K, using both an 8 A and a 10 A force-shifted nonbonded cutoff. The ensemble averages of both simulations are in reasonable agreement with the experimental helical content from circular dichroism (CD), the (3)J(HNalpha) coupling constants, and the 57 observed NOEs. Analysis of the structures from both simulations revealed very little formation of contiguous i --> i + 3 hydrogen bonds (3(10)-helix); however, there was a large population of bifurcated i --> i + 3 and i --> i + 4 alpha-helical hydrogen bonds. In addition, both simulations contained considerable populations of pi-helix (i --> i + 5 hydrogen bonds). Individual turns formed over residues 1-9, which we predict contribute to the intensities of the experimentally observed alphaN(i,i + 2) NOEs. Here we show how sampling of both folded and unfolded structures can provide a structural framework for deconvolution of the conformational contributions to experimental ensemble averages.

Elastin undergoes an inverse temperature transition and collapses at high temperatures in both simulation and experiment. We investigated a pH-dependent modification of this transition by simulating a glutamic acid (Glu)-substituted elastin at varying pHs and temperatures. The Glu-substituted peptide collapsed at higher temperature than the unsubstituted elastin when Glu was charged. The charge effects could be reversed by neutralization of the Glu carboxyl groups at low pH, and in that case the peptide collapsed at a lower temperature. The collapse was accompanied by the formation of beta-turns and short distorted beta-sheets. Formation of contacts between hydrophobic side chains drives the collapse at high temperature, but interactions between water and polar groups (Glu and main chain) can attenuate this effect at high pH. The overall competition and balance of the polar and nonpolar groups determined the conformational states of the peptide. Water hydration contributed to the conformational transition, and the peptide and its hydration shell must be considered. Structurally, waters near polar residues mainly formed hydrogen bonds with the protein atoms, while waters around the hydrophobic side chains tended to be parallel to the peptide groups to maximize water-water interactions., (Copyright 2002 Wiley Periodicals, Inc. Biopolymers 68: 121-129, 2003)

The structure and dynamics of the water hydrating peptides and proteins are examined here at atomic resolution via molecular dynamics simulations. Detailed solvation density and residence time data for all 20 L-amino acids in an end-capped AXA tripeptide motif are presented. In addition, the solvation of the protein chymotrypsin inhibitor 2 is investigated as a point of comparison. Residues on the surface of proteins are not isolated; they interact both locally and non-locally in sequence space, and comparison of the solvation properties of each amino acid in both the peptide and protein allow us to distinguish inherent solvation properties from context-dependent perturbations due to neighboring residues. This work moves beyond traditional radial distribution functions and presents graphical representations of preferential solvation and orientation of water by side chains and the main chain. The combination of 0.3 micros of simulation data improves the statistical sampling over previous studies and reveals the significance of bridging water molecules that stabilize and mediate side chain-side chain, side chain-main chain and main chain-main chain interactions at the solvation interface.

We have traditionally relied on extremely elevated temperatures (498K, 225 degrees C) to investigate the unfolding process of proteins within the timescale available to molecular dynamics simulations with explicit solvent. However, recent advances in computer hardware have allowed us to extend our thermal denaturation studies to much lower temperatures. Here we describe the results of simulations of chymotrypsin inhibitor 2 at seven temperatures, ranging from 298K to 498K. The simulation lengths vary from 94ns to 20ns, for a total simulation time of 344ns, or 0.34 micros. At 298K, the protein is very stable over the full 50ns simulation. At 348K, corresponding to the experimentally observed melting temperature of CI2, the protein unfolds over the first 25ns, explores partially unfolded conformations for 20ns, and then refolds over the last 35ns. Above its melting temperature, complete thermal denaturation occurs in an activated process. Early unfolding is characterized by sliding or breathing motions in the protein core, leading to an unfolding transition state with a weakened core and some loss of secondary structure. After the unfolding transition, the core contacts are rapidly lost as the protein passes on to the fully denatured ensemble. While the overall character and order of events in the unfolding process are well conserved across temperatures, there are substantial differences in the timescales over which these events take place. We conclude that 498K simulations are suitable for elucidating the details of protein unfolding at a minimum of computational expense.

To investigate whether swapping native turns of a globular protein with an elastin-based turn sequence (VPGVG) can increase its thermostability, we have performed molecular dynamics simulations of wild-type chymotrypsin inhibitor 2 (CI2) and variants containing elastin-based turns at 10 degrees C and 40 degrees C. Wild-type CI2 is more stable at 10 degrees C, while both of the variant forms are more stable at 40 degrees C. Detailed analyses indicate that the elastin-based turns do indeed contribute to the inverse temperature behavior of the modified proteins. Therefore, swapping a wild-type turn sequence with an elastin-based turn provides a novel way to both improve stability of target proteins at body temperature and to possibly introduce a temperature-sensitive switch.

Uropathogenic Escherichia coli account for the largest proportion of nosocomial infections in the United States. Nosocomial infections are a major source of increased costs and treatment complications. Many infections are biofilm associated, rendering antibiotic treatments ineffective or cause additional complications (e.g., microbiome depletion). This work presents a potentially complementary non-antibiotic strategy to fight nosocomial infections by inhibiting the formation of amyloid fibrils, a proteinaceous structural reinforcement known as curli in E. coli biofilms. Despite extensive characterization of the fibrils themselves and their associated secretion system, mechanistic details of curli assembly in vivo remain unclear. We hypothesized that, like other amyloid fibrils, curli polymerization involves a unique secondary structure termed "α-sheet". Biophysical studies herein confirmed the presence of α-sheet structure in prefibrillar species of CsgA, the major component of curli, as it aggregated. Binding of synthetic α-sheet peptides to the soluble α-sheet prefibrillar species inhibited CsgA aggregation in vitro and suppressed amyloid fibril formation in biofilms. Application of synthetic α-sheet peptides also enhanced antibiotic susceptibility and dispersed biofilm-resident bacteria for improved uptake by phagocytic cells. The ability of synthetic α-sheet peptides to reduce biofilm formation, improve antibiotic susceptibility, and enhance clearance by macrophages has broad implications for combating biofilm-associated infections. [ABSTRACT FROM AUTHOR]

Abstract: Small β-hairpin peptides have been widely used as models for the folding of β-sheets. But how applicable is the folding of such models to β-structure in larger proteins with conventional hydrophobic cores? Here we present multiple unfolding simulations of three such proteins that contain the WW domain double hairpin β-sheet motif: cold shock protein A (CspA), cold shock protein B (CspB) and glucose permease IIA domain. We compare the behavior of the free motif in solution and in the context of proteins of different size and architecture. Both Csp proteins lost contacts between the double-hairpin motif and the protein core as the first step of unfolding and proceeded to unfold with loss of the third β-strand, similar to the isolated WW domain. The glucose permease IIA domain is a larger protein and the contacts between the motif and the core were not lost as quickly. Instead the unfolding pathway of glucose permease IIA followed a different pathway with β1 pulling away from the sheet first. Interestingly, when the double hairpin motif was excised from the glucose permease IIA domain and simulated in isolation in water it unfolded by the same pathway as the WW domain, indicating that it is tertiary interactions with the protein that alter the motif’s unfolding not a sequence dependent effect on its intrinsic unfolding behavior. With respect to the unfolding of the hairpins, there was no consistent order to the loss of hydrogen bonds between the β-strands in the hairpins in any of the systems. Our results show that while the folding behavior of the isolated WW domain is generally consistent with the double hairpin motif’s behavior in the cold shock proteins, it is not the case for the glucose permease IIA domain. So, one must be cautious in extrapolating findings from model systems to larger more complicated proteins where tertiary interactions can overwhelm intrinsic behavior. [Copyright &y& Elsevier]

Members of the homeodomain superfamily are three-helix bundle proteins whose second and third helices form a helix-turn-helix motif (HTH). Their folding mechanism slides from the ultrafast, three-state framework mechanism for the engrailed homeodomain (EnHD), in which the HTH motif is independently stable, to an apparent two-state nucleation-condensation, model for family members with an unstable HTH motif. The folding intermediate of EnHO has nearly native HTH structure, but it is not docked with helixi. The determinant of whether twoor three-state folding was hypothesized to be the stability of the HTH substructure. Here, we describe a detailed 0-value analysis of the folding of the Piti homeodomain, which has similar ultrafast kinetics to that of EnHD. Formation of helixi was strongly coupled with formation of HTH, which was initially surprising because they are uncoupled in the EnHD folding intermediate. However, we found a key difference between Piti and EnHD: The isolated peptide corresponding to the HTH motif in Piti was not folded in the absence of Hi. Independent molecular dynamics simulations of Piti unfolding found an intermediate with Hi misfolded onto the HTH motif. The Piti folding pathway is the connection between that of EnHD and the slower folding homeodomains and provides a link in the transition of mechanisms from twoto three-state folding in this superfamily. The malleability of folding intermediates can lead to unstable substructures being stabilized by a variety of nonnative interactions, adding to the continuum of folding mechanisms. [ABSTRACT FROM AUTHOR]

Here, we compare the distributions of main chain (ΦΨ) angles (i.e., Ramachandran maps) of the 20 naturally occurring amino acids in three contexts: (i) molecular dynamics (MD) simulations of Gly-Gly-X-Gly-Gly pentapeptides in water at 298 K with exhaustive sampling, where X = the amino acid in question; (ii) 188 independent protein simulations in water at 298 K from our Dynameomics Project; and (iii) static crystal and NMR structures from the Protein Data Bank. The GGXGG peptide series is often used as a model of the unstructured denatured state of proteins. The sampling in the peptide MD simulations is neither random nor uniform. Instead, individual amino acids show preferences for particular conformations, but the peptide is dynamic, and interconversion between conformers is facile. For a given amino acid, the (ΦΨ) distributions in the protein simulations and the Protein Data Bank are very similar and often distinct from those in the peptide simulations. Comparison between the peptide and protein simulations shows that packing constraints, solvation. and the tendency for particular amino acids to be used for specific structural motifs can overwhelm the "intrinsic propensities" of amino acids for particular (4,'I') conformations. We also compare our helical propensities with experimental consensus values using the host-guest method, which appear to be determined largely by context and not necessarily the intrinsic conformational propensities of the guest residues. These simulations represent an improved coil library free from contextual effects to better model intrinsic conformational propensities and provide a detailed view of conformations making up the "random coil" state. [ABSTRACT FROM AUTHOR]

The article discusses various reports published within the issue, including one on multi-domain protein misfolding, another by Dan Raleigh on the role of helical structures in amyloid formation by a number of peptides, and another by Van der Kamp et al on mutations associated with human prion diseases.

The homotetramer of transthyretin (TTR) dissociates into a monomeric amyloidogenic intermediate that self-assembles into amyloid fibrils at low pH. We have performed molecular dynamics simulations of monomeric TTR at neutral and low pH at physiological (310 K) and very elevated temperature (498 K). In the low-pH simulations at both temperatures, one of the two β-sheets (strands CBEF) becomes disrupted, and α-sheet structure forms in the other sheet (strands DAGH). α-sheet is formed by alternating αL and αR residues, and it was first proposed by Pauling and Corey. Overall, the simulations are in agreement with the available experimental observations, including solid-state NMR results for a TTR-peptide amyloid. In addition, they provide a unique explanation for the results of hydrogen exchange experiments of the amyloidogenic intermediate—results that are difficult to explain with β-structure. We propose that α-sheet may represent a key pathological conformation during amyloidogenesis. [Copyright &y& Elsevier]

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