Association of Helical β-Peptides and their Aggregation Behavior from the Potential of Mean Force in Explicit Solvent

Helical β-peptides have been shown to fold into well-defined structures. In aqueous solution, some β-peptides self-assemble into nanoscale fibers, aggregates, and liquid crystalline phases. Molecular simulations, at an atomistic level, are used to examine, in a systematic manner, the interactions between distinct β-peptide molecules. The relationship between side-chain chemistry (and position along the backbone) and, in particular, aggregation behaviors, is assessed by calculating the potential of mean force or dimerization free energy of two peptides in explicit water. The free energy profiles as a function of separation for helical, amphiphilic β-peptides are consistent with experimental observations, and help explain the origins of aggregate or fiber formation in solution. Close examination of the energetic and entropic contributions to the free energy reveals that, depending on the position of certain side groups along the molecule, the tendency of two peptides to aggregate can be driven by entropy or by energy, respectively. In contrast to findings from previous works that employed a coarse representation of the solvent, it is shown that water-peptide interactions play key roles in the association behavior of β-peptides.     

Conformational sampling with implicit solvent models: application to the PHF6 peptide in tau protein

Implicit solvent models approximate the effects of solvent through a potential of mean force and therefore make solvated simulations computationally efficient. Yet despite their computational efficiency, the inherent approximations made by implicit solvent models can sometimes lead to inaccurate results. To test the accuracy of a number of popular implicit solvent models, we determined whether implicit solvent simulations can reproduce the set of potential energy minima obtained from explicit solvent simulations. For these studies, we focus on a six-residue amino-acid sequence, referred to as the paired helical filament 6 (PHF6), which may play an important role in the formation of intracellular aggregates in patients with Alzheimer's disease. Several implicit solvent models form the basis of this work--two based on the generalized Born formalism, and one based on a Gaussian solvent-exclusion model. All three implicit solvent models generate minima that are in good agreement with minima obtained from simulations with explicit solvent. Moreover, free-energy profiles generated with each implicit solvent model agree with free-energy profiles obtained with explicit solvent. For the Gaussian solvent-exclusion model, we demonstrate that a straightforward ranking of the relative stability of each minimum suggests that the most stable structure is extended, a result in excellent agreement with the free-energy profiles. Overall, our data demonstrate that for some peptides like PHF6, implicit solvent can accurately reproduce the set of local energy minimum arising from quenched dynamics simulations with explicit solvent. More importantly, all solvent models predict that PHF6 forms extended beta-structures in solution, a finding consistent with the notion that PHF6 initiates neurofibrillary tangle formation in patients with Alzheimer's disease.     

Thermodynamic stability of beta-peptide helices and the role of cyclic residues

Beta-peptides are emerging as an attractive class of peptidomimetic molecules. In contrast to naturally occurring alpha-peptides, short oligomers of beta-amino acids (comprising just 4-6 monomers) exhibit stable secondary structures that make them amenable for quantitative, concerted experimental and theoretical studies of the effects of particular chemical interactions on structure. In this work, molecular simulations are used to study the thermodynamic stability of helical conformations formed by beta-peptides containing varying proportions of acyclic (beta(3)) and cyclic (ACH) residues. More specifically, several beta-peptides differing only in their content of cyclic residues are considered in this work. Previous computational studies of beta-peptides have relied mostly on energy minimization of molecular dynamics simulations. In contrast, our study relies on density-of-states based Monte Carlo simulations to calculate the free energy and examine the stability of various folded structures of these molecules along a well-defined order parameter. By resorting to an expanded-ensemble formalism, we are able to determine the free energy required to unfold specific molecules, a quantity that could be measured directly through single-molecule force spectroscopy. Simulations in both implicit and explicit solvents have permitted a systematic study of the role of cyclic residues and electrostatics on the stability of secondary structures. The molecules considered in this work are shown to exhibit stable H-14 helical conformations and, in some cases, relatively stable H-12 conformations, thereby suggesting that solvent quality may be used to manipulate the hydrogen-bonding patterns and structure of these peptides.     

A comparison of the different helices adopted by ��- and ��-peptides suggests different reasons for their stability

The right‐handed α‐helix is the dominant helical fold of α‐peptides, whereas the left‐handed 3₁₄‐helix is the dominant helical fold of β‐peptides. Using molecular dynamics simulations, the properties of α‐helical α‐peptides and 3₁₄‐helical β‐peptides with different C‐terminal protonation states and in the solvents water and methanol are compared. The observed energetic and entropic differences can be traced to differences in the polarity of the solvent‐accessible surface area and, in particular, the solute dipole moments, suggesting different reasons for their stability.     

Spontaneous transmembrane pore formation by short-chain synthetic peptide

Amphiphilic β-peptides, which are synthetically designed short-chain helical foldamers of β-amino acids, are established potent biomimetic alternatives of natural antimicrobial peptides. An intriguing question is how the distinct molecular architecture of these short-chain and rigid synthetic peptides translates to its potent membrane-disruption ability. Here, we address this question via a combination of all-atom and coarse-grained molecular dynamics simulations of the interaction of mixed phospholipid bilayer with an antimicrobial 10-residue globally amphiphilic helical β-peptide at a wide range of concentrations. The simulation demonstrates that multiple copies of this synthetic peptide, initially placed in aqueous solution, readily self-assemble and adsorb at membrane interface. Subsequently, beyond a threshold peptide/lipid ratio, the surface-adsorbed oligomeric aggregate moves inside the membrane and spontaneously forms stable water-filled transmembrane pores via a cooperative mechanism. The defects induced by these pores lead to the dislocation of interfacial lipid headgroups, membrane thinning, and substantial water leakage inside the hydrophobic core of the membrane. A molecular analysis reveals that despite having a short architecture, these synthetic peptides, once inside the membrane, would stretch themselves toward the distal leaflet in favor of potential contact with polar headgroups and interfacial water layer. The pore formed in coarse-grained simulation was found to be resilient upon structural refinement. Interestingly, the pore-inducing ability was found to be elusive in a non-globally amphiphilic sequence isomer of the same β-peptide, indicating strong sequence dependence. Taken together, this work puts forward key perspectives of membrane activity of minimally designed synthetic biomimetic oligomers relative to the natural antimicrobial peptides.     

Preparation and conformational analysis of C-glycosyl β- and β/β-peptides

Ten C-glycosyl β²- and β/β²-peptides with three to eight amino acid residues have been prepared. Solution and solid-phase peptide syntheses were employed to assemble β²-amino acids in which C-glycosylic substituents are attached to the C-2 position of β-amino acids. Conformational analysis of the C-glycosyl β²-peptides using NMR and CD spectra indicates that the tripeptide can form a helical secondary structure. Besides, helix directions of the C-glycosyl β/β²-peptides are governed by the configuration at the α-carbon of the peptide backbone that originates from the stereocenter of the C-glycosyl β²-amino acids.     

Synthesis of peptides and glycopeptides with polyproline II helical topology as potential antifreeze molecules

A library of peptides and glycopeptides containing (4R)-hydroxy-l-proline (Hyp) residues were designed with a view to providing stable polyproline II (PPII) helical molecules with antifreeze activity. A library of dodecapeptides containing contiguous Hyp residues or an Ala-Hyp-Ala tripeptide repeat sequence were synthesized with and without α-O-linked N-acetylgalactosamine and α-O-linked galactose-β-(1→3)-N-acetylgalactosamine appended to the peptide backbone. All (glyco)peptides possessed PPII helical secondary structure with some showing significant thermal stability. The majority of the (glyco)peptides did not exhibit thermal hysteresis (TH) activity and were not capable of modifying the morphology of ice crystals. However, an unglycosylated Ala-Hyp-Ala repeat peptide did show significant TH and ice crystal re-shaping activity suggesting that it was capable of binding to the surface of ice. All (glyco)peptides synthesized displayed some ice recrystallization inhibition (IRI) activity with unglycosylated peptides containing the Ala-Hyp-Ala motif exhibiting the most potent inhibitory activity. Interestingly, although glycosylation is critical to the activity of native antifreeze glycoproteins (AFGPs) that possess an Ala-Thr-Ala tripeptide repeat, this same structural modification is detrimental to the antifreeze activity of the Ala-Hyp-Ala repeat peptides studied here.     

Evaluating Molecular Mechanical Potentials for Helical Peptides and Proteins

Multiple variants of the AMBER all-atom force field were quantitatively evaluated with respect to their ability to accurately characterize helix-coil equilibria in explicit solvent simulations. Using a global distributed computing network, absolute conformational convergence was achieved for large ensembles of the capped A₂₁ and F_s helical peptides. Further assessment of these AMBER variants was conducted via simulations of a flexible 164-residue five-helix-bundle protein, apolipophorin-III, on the 100 ns timescale. Of the contemporary potentials that had not been assessed previously, the AMBER-99SB force field showed significant helix-destabilizing tendencies, with beta bridge formation occurring in helical peptides, and unfolding of apolipophorin-III occurring on the tens of nanoseconds timescale. The AMBER-03 force field, while showing adequate helical propensities for both peptides and stabilizing apolipophorin-III, (i) predicts an unexpected decrease in helicity with ALA→ARG⁺ substitution, (ii) lacks experimentally observed 3₁₀ helical content, and (iii) deviates strongly from average apolipophorin-III NMR structural properties. As is observed for AMBER-99SB, AMBER-03 significantly overweighs the contribution of extended and polyproline backbone configurations to the conformational equilibrium. In contrast, the AMBER-99φ force field, which was previously shown to best reproduce experimental measurements of the helix-coil transition in model helical peptides, adequately stabilizes apolipophorin-III and yields both an average gyration radius and polar solvent exposed surface area that are in excellent agreement with the NMR ensemble.     

Folding thermodynamics of β‐hairpins studied by replica‐exchange molecular dynamics simulations

We study the differences in folding stability of β‐hairpin peptides, including GB1 hairpin and a point mutant GB1 K10G, as well as tryptophan zippers (TrpZips): TrpZip1, TrpZip2, TrpZip3‐1, and TrpZip4. By performing replica‐exchange molecular dynamics simulations with Amber03* force field (a modified version of Amber ff03) in explicit solvent, we observe ab initio folding of all the peptides except TrpZip3‐1, which is experimentally known to be the least stable among the peptides studied here. By calculating the free energies of unfolding of the peptides at room temperature and folding midpoint temperatures for thermal unfolding of peptides, we find that TrpZip4 and GB1 K10G peptides are the most stable β‐hairpins followed by TrpZip1, GB1, and TrpZip2 in the given order. Hence, the proposed K10G mutation of GB1 peptide results in enhanced stability compared to wild‐type GB1. An important goal of our study is to test whether simulations with Amber 03* model can reproduce experimentally predicted folding stability differences between these peptides. While the stabilities of GB1 and TrpZip1 yield close agreement with experiment, TrpZip2 is found to be less stable than predicted by experiment. However, as heterogenous folding of TrpZip2 may yield divergent thermodynamic parameters by different spectroscopic methods, mismatching of results with previous experimental values are not conclusive of model shortcomings. For most of the cases, molecular simulations with Amber03* can successfully reproduce experimentally known differences between the mutated peptides, further highlighting the predictive capabilities of current state‐of‐the‐art all‐atom protein force fields. Proteins 2015; 83:1307–1315. © 2015 Wiley Periodicals, Inc.     

The amyloid precursor protein forms plasmalemmal clusters via its pathogenic amyloid-β domain

The amyloid precursor protein (APP) is a large, ubiquitous integral membrane protein with a small amyloid-β (Aβ) domain. In the human brain, endosomal processing of APP produces neurotoxic Aβ-peptides, which are involved in Alzheimer's disease. Here, we show that the Aβ sequence exerts a physiological function when still present in the unprocessed APP molecule. From the extracellular site, Aβ concentrates APP molecules into plasmalemmal membrane protein clusters. Moreover, Aβ stabilization of clusters is a prerequisite for their targeting to endocytic clathrin structures. Therefore, we conclude that the Aβ domain directly mediates a central step in APP trafficking, driving its own conversion into neurotoxic peptides.     

Synthesis and characterization of cyclic peptides that are β‐helical in trifluoroethanol

We show that three designed cyclic d ,l ‐peptides are β‐helical in TFE—a solvent in which the archetypal β‐helical peptide, gA, is unstructured. This result represents an advance in the field of β‐helical peptide foldamers and a step toward achieving β‐helical structure under a broad range of solvent conditions. We synthesized two of the three peptides examined using an improved variant of our original CBC strategy. Here, we began with a commercially available PEG–PS composite resin prefunctionalized with the alkanesulfonamide ‘SCL’ linker and preloaded with glycine. Our new conditions avoided C‐terminal epimerization during the CBC step and simplified purification. In addition, we present results to define the scope and limitations of our CBC strategy. These methods and observations will prove useful in designing additional cyclic β‐helical peptides for applications ranging from transmembrane ion channels to ligands for macromolecular targets. Published 2014. This article is a U.S. Government work and is in the public domain in the USA.     

Tackling Force-Field Bias in Protein Folding Simulations: Folding of Villin HP35 and Pin WW Domains in Explicit Water

The ability to fold proteins on a computer has highlighted the fact that existing force fields tend to be biased toward a particular type of secondary structure. Consequently, force fields for folding simulations are often chosen according to the native structure, implying that they are not truly “transferable.” Here we show that, while the AMBER ff03 potential is known to favor helical structures, a simple correction to the backbone potential (ff03^∗) results in an unbiased energy function. We take as examples the 35-residue α-helical Villin HP35 and 37 residue β-sheet Pin WW domains, which had not previously been folded with the same force field. Starting from unfolded configurations, simulations of both proteins in Amber ff03^∗ in explicit solvent fold to within 2.0 Å RMSD of the experimental structures. This demonstrates that a simple backbone correction results in a more transferable force field, an important requirement if simulations are to be used to interpret folding mechanism.     

Secondary Structure Propensities in Peptide Folding Simulations: A Systematic Comparison of Molecular Mechanics Interaction Schemes

We present a systematic study directed toward the secondary structure propensity and sampling behavior in peptide folding simulations with eight different molecular dynamics force-field variants in explicit solvent. We report on the combinational result of force field, water model, and electrostatic interaction schemes and compare to available experimental characterization of five studied model peptides in terms of reproduced structure and dynamics. The total simulation time exceeded 18 μs and included simulations that started from both folded and extended conformations. Despite remaining sampling issues, a number of distinct trends in the folding behavior of the peptides emerged. Pronounced differences in the propensity of finding prominent secondary structure motifs in the different applied force fields suggest that problems point in particular to the balance of the relative stabilities of helical and extended conformations.     

Probing Energetics of Aβ Fibril Elongation by Molecular Dynamics Simulations

Using replica exchange molecular dynamics simulations and an all-atom implicit solvent model, we probed the energetics of Aβ_10-40 fibril growth. The analysis of the interactions between incoming Aβ peptides and the fibril led us to two conclusions. First, considerable variations in fibril binding propensities are observed along the Aβ sequence. The peptides in the fibril and those binding to its edge interact primarily through their N-termini. Therefore, the mutations affecting the Aβ positions 10-23 are expected to have the largest impact on fibril elongation compared with those occurring in the C-terminus and turn. Second, we performed weak perturbations of the binding free energy landscape by scanning partial deletions of side-chain interactions at various Aβ sequence positions. The results imply that strong side-chain interactions—in particular, hydrophobic contacts—impede fibril growth by favoring disordered docking of incoming peptides. Therefore, fibril elongation may be promoted by moderate reduction of Aβ hydrophobicity. The comparison with available experimental data is presented.     

Helix stability of oligoglycine,oligoalanine, and oligo‐β‐alanine dodecamers reflected by hydrogen‐bond persistence

Helices are important structural/recognition elements in proteins and peptides. Stability and conformational differences between helices composed of α‐ and β‐amino acids as scaffolds for mimicry of helix recognition has become a theme in medicinal chemistry. Furthermore, helices formed by β‐amino acids are experimentally more stable than those formed by α‐amino acids. This is paradoxical because the larger sizes of the hydrogen‐bonding rings required by the extra methylene groups should lead to entropic destabilization. In this study, molecular dynamics simulations using the second‐generation force field, AMOEBA (Ponder, J.W., et al., Current status of the AMOEBA polarizable force field. J Phys Chem B, 2010. 114 (8): p. 2549–64.) explored the stability and hydrogen‐bonding patterns of capped oligo‐β‐alanine, oligoalanine, and oligoglycine dodecamers in water. The MD simulations showed that oligo‐β‐alanine has strong acceptor+2 hydrogen bonds, but surprisingly did not contain a large content of 3₁₂‐helical structures, possibly due to the sparse distribution of the 3₁₂‐helical structure and other structures with acceptor+2 hydrogen bonds. On the other hand, despite its backbone flexibility, the β‐alanine dodecamer had more stable and persistent <3.0 Å hydrogen bonds. Its structure was dominated more by multicentered hydrogen bonds than either oligoglycine or oligoalanine helices. The 3₁ (PII) helical structure, prevalent in oligoglycine and oligoalanine, does not appear to be stable in oligo‐β‐alanine indicating its competition with other structures (stacking structure as indicated by MD analyses). These differences are among the factors that shape helical structural preferences and the relative stabilities of these three oligopeptides. Proteins 2014; 82:3043–3061. © 2014 Wiley Periodicals, Inc.     

Dimerization of helical β-peptides in solution

Molecular simulations are used to examine the aggregation behavior of several β-peptides in explicit water. The particular peptides considered here adopt a helical, rodlike conformation in aqueous solution. Four distinct molecular sequences are considered. Earlier experimental studies have revealed the formation of ordered and disordered aggregates for such molecules, depending on sequence. The simulations reported here, which are conducted by resorting to metadynamics techniques, lead to free energy surfaces for dimerization of the peptides in water as a function of separation and relative orientation. Such surfaces are used to identify the molecular origins for the behaviors observed in the experiments.     

Bacterial species selective toxicity of two isomeric α/β-peptides: Role of membrane lipids

We have studied how membrane interactions of two synthetic cationic antimicrobial peptides with alternating α- and β-amino acid residues (“α/β-peptides”) impact toxicity to different prokaryotes. Electron microscopic examination of thin sections of Escherichia coli and of Bacillus subtilis exposed to these two α/β-peptides reveals different structural changes in the membranes of these bacteria. These two peptides also have very different effects on the morphology of liposomes composed of phosphatidylethanolamine and phosphatidylglycerol in a 2:1 molar ratio. Freeze fracture electron microscopy indicates that with this lipid mixture, α/β-peptide I induces the formation of a sponge phase. ³¹P NMR and X-ray diffraction are consistent with this conclusion. In contrast, with α/β-peptide II and this same lipid mixture, a lamellar phase is maintained, but with a drastically reduced d-spacing. α/β-Peptide II is more lytic to liposomes composed of these lipids than is I. These findings are consistent with the greater toxicity of α/β-peptide II, relative to α/β-peptide I, to E. coli, a bacterium having a high content of phosphatidylethanolamine. In contrast, both α/β-peptides display similar toxicity toward B. subtilis, in accord with the greater anionic lipid composition in its membrane. This work shows that variations in the selectivity of these peptidic antimicrobial peptides toward different strains of bacteria can be partly determined by the lipid composition of the bacterial cell membrane.     

Mapping Conformational Ensembles of Aβ Oligomers in Molecular Dynamics Simulations

Although the oligomers formed by Aβ peptides appear to be the primary cytotoxic species in Alzheimer's disease, detailed information about their structures appears to be lacking. In this article, we use exhaustive replica exchange molecular dynamics and an implicit solvent united-atom model to study the structural properties of Aβ monomers, dimers, and tetramers. Our analysis suggests that the conformational ensembles of Aβ dimers and tetramers are very similar, but sharply distinct from those sampled by the monomers. The key conformational difference between monomers and oligomers is the formation of β-structure in the oligomers occurring together with the loss of intrapeptide interactions and helix structure. Our simulations indicate that, independent of oligomer order, the Aβ aggregation interface is largely confined to the sequence region 10-23, which forms the bulk of interpeptide interactions. We show that the fractions of β structure computed in our simulations and measured experimentally are in good agreement.     

Replica Exchange Simulations of the Thermodynamics of Aβ Fibril Growth

Replica exchange molecular dynamics and an all-atom implicit solvent model are used to probe the thermodynamics of deposition of Alzheimer's Aβ monomers on preformed amyloid fibrils. Consistent with the experiments, two deposition stages have been identified. The docking stage occurs over a wide temperature range, starting with the formation of the first peptide-fibril interactions at 500 K. Docking is completed when a peptide fully adsorbs on the fibril edge at the temperature of 380 K. The docking transition appears to be continuous, and occurs without free energy barriers or intermediates. During docking, incoming Aβ monomer adopts a disordered structure on the fibril edge. The locking stage occurs at the temperature of ≈360 K and is characterized by the rugged free energy landscape. Locking takes place when incoming Aβ peptide forms a parallel β-sheet structure on the fibril edge. Because the β-sheets formed by locked Aβ peptides are typically off-registry, the structure of the locked phase differs from the structure of the fibril interior. The study also reports that binding affinities of two distinct fibril edges with respect to incoming Aβ peptides are different. The peptides bound to the concave edge have significantly lower free energy compared to those bound on the convex edge. Comparison with the available experimental data is discussed.     

Probing the Conformation of FhaC with Small-Angle Neutron Scattering and Molecular Modeling

Probing the solution structure of membrane proteins represents a formidable challenge, particularly when using small-angle scattering. Detergent molecules often present residual scattering contributions even at their match point in small-angle neutron scattering (SANS) measurements. Here, we studied the conformation of FhaC, the outer-membrane, β-barrel transporter of the Bordetella pertussis filamentous hemagglutinin adhesin. SANS measurements were performed on homogeneous solutions of FhaC solubilized in n-octyl-d17-βD-glucoside and on a variant devoid of the α helix H1, which critically obstructs the FhaC pore, in two solvent conditions corresponding to the match points of the protein and the detergent, respectively. Protein-bound detergent amounted to 142 ± 10 mol/mol as determined by analytical ultracentrifugation. By using molecular modeling and starting from three distinct conformations of FhaC and its variant embedded in lipid bilayers, we generated ensembles of protein-detergent arrangement models with 120–160 detergent molecules. The scattered curves were back-calculated for each model and compared with experimental data. Good fits were obtained for relatively compact, connected detergent belts, which occasionally displayed small detergent-free patches on the outer surface of the β barrel. The combination of SANS and modeling clearly enabled us to infer the solution structure of FhaC, with H1 inside the pore as in the crystal structure. We believe that our strategy of combining explicit atomic detergent modeling with SANS measurements has significant potential for structural studies of other detergent-solubilized membrane proteins.