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During amyloidogenesis, proteins undergo conformational changes that allow them to aggregate and assemble into insoluble, fibrillar structures. Soluble oligomers that form during this process typically contain 2–24 monomeric subunits and are cytotoxic. Before the formation of these soluble oligomers, monomeric species first adopt aggregation-competent conformations. Knowledge of the structures of these intermediate states is invaluable to the development of molecular strategies to arrest pathological amyloid aggregation. However, the highly dynamic and interconverting nature of amyloidogenic species limits biophysical characterization of their structures during amyloidogenesis. Here, we use molecular dynamics simulations to probe conformations sampled by monomeric transthyretin under amyloidogenic conditions. We show that certain β-strands in transthyretin tend to unfold and sample nonnative conformations and that the edge strands in one β-sheet (the DAGH sheet) are particularly susceptible to conformational changes in the monomeric state. We also find that changes in the tertiary structure of transthyretin can be associated with disruptions to the secondary structure. We evaluated the conformations produced by molecular dynamics by calculating how well molecular-dynamics-derived structures reproduced NMR-derived interatomic distances. Finally, we leverage our computational results to produce experimentally testable hypotheses that may aid experimental explorations of pathological conformations of transthyretin.

Amyloid diseases make up a set of fatal disorders in which proteins aggregate to form fibrils that deposit in tissues throughout the body. Amyloid-associated diseases are challenging to study because amyloid formation occurs on time scales that span several orders of magnitude and involve heterogeneous, interconverting protein conformations. The development of more effective technologies to diagnose and treat amyloid disease requires both a map of the conformations sampled during amyloidogenesis and an understanding of the molecular mechanisms that drive this process. In prior molecular dynamics simulations of amyloid proteins, we observed the formation of a nonstandard type of secondary structure, called α-sheet, that we proposed is associated with the pathogenic conformers in amyloid disease, the soluble oligomers. However, the detailed molecular interactions that drive the conversion to α-sheet remain elusive. Here we use molecular dynamics simulations to interrogate a critical event in transthyretin aggregation, the formation of aggregation-competent, monomeric species. We show that conformational changes in one of the two β-sheets in transthyretin enable solvent molecules and polar side chains to form electrostatic interactions with main-chain peptide groups to facilitate and modulate conversion to α-sheet secondary structure. Our results shed light on the early conformational changes that drive transthyretin toward the α-sheet structure associated with toxicity. Delineation of the molecular events that lead to aggregation at atomic resolution can aid strategies to target the early, critical toxic soluble oligomers.

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.

Various host-guest peptide series are used by experimentalists as reference conformational states. One such use is as a baseline for random-coil NMR chemical shifts. Comparison to this random-coil baseline, through secondary chemical shifts, is used to infer protein secondary structure. The use of these random-coil data sets rests on the perception that the reference chemical shifts arise from states where there is little or no conformational bias. However, there is growing evidence that the conformational composition of natively and nonnatively unfolded proteins fail to approach anything that can be construed as random coil. Here, we use molecular dynamics simulations of an alanine-based host-guest peptide series (AAXAA) as a model of unfolded and denatured states to examine the intrinsic propensities of the amino acids. We produced ensembles that are in good agreement with the experimental NMR chemical shifts and confirm that the sampling of the 20 natural amino acids in this peptide series is be far from random. Preferences toward certain regions of conformational space were both present and dependent upon the environment when compared under conditions typically used to denature proteins, i.e., thermal and chemical denaturation. Moreover, the simulations allowed us to examine the conformational makeup of the underlying ensembles giving rise to the ensemble-averaged chemical shifts. We present these data as an intrinsic backbone propensity library that forms part of our Structural Library of Intrinsic Residue Propensities to inform model building, to aid in interpretation of experiment, and for structure prediction of natively and nonnatively unfolded states.

Prion diseases involve the conformational conversion of the cellular prion protein (PrPC) to its misfolded pathogenic form (PrPSc). To better understand the structural mechanism of this conversion, we performed extensive all-atom, explicit-solvent molecular-dynamics simulations for three structures of the wild-type human PrP (huPrP) at different pH values and temperatures. Residue 129 is polymorphic, being either Met or Val. Two of the three structures have Met in position 129 and the other has Val. Lowering the pH or raising the temperature induced large conformational changes of the C-terminal globular domain and increased exposure of its hydrophobic core. In some simulations, HA and its preceding S1-HA loop underwent large displacements. The C-terminus of HB was unstable and sometimes partially unfolded. Two hydrophobic residues, Phe-198 and Met-134, frequently became exposed to solvent. These conformational changes became more dramatic at lower pH or higher temperature. Furthermore, Tyr-169 and the S2-HB loop, or the X-loop, were different in the starting structures but converged to common conformations in the simulations for the Met-129, but not the Val-129, protein. α-Strands and β-strands formed in the initially unstructured N-terminus. α-Strand propensity in the N-terminus was different between the Met-129 and Val129 proteins, but β-strand propensity was similar. This study reveals detailed structural and dynamic properties of huPrP, providing insight into the mechanism of the conversion of PrPCto PrPSc.

Prion diseases are caused by misfolding and aggregation of the prion protein (PrP). Pathogenic mutations such as Y218N and E196K are known to cause Gerstmann-Sträussler-Scheinker syndrome and Creutzfeldt-Jakob disease, respectively. Here we describe molecular dynamics simulations of these mutant proteins to better characterize the detailed conformational effects of these sequence substitutions. Our results indicate that the mutations disrupt the wild-type native PrPCstructure and cause misfolding. Y218N reduced hydrophobic packing around the X-loop (residues 165–171), and E196K abolished an important wild-type salt bridge. While differences in the mutation site led PrP mutants to misfold along different pathways, we observed multiple traits of misfolding that were common to both mutants. Common traits of misfolding included: 1) detachment of the short helix (HA) from the PrP core; 2) exposure of side chain F198; and 3) formation of a nonnative strand at the N-terminus. The effect of the E196K mutation directly abolished the wild-type salt bridge E196-R156, which further destabilized the F198 hydrophobic pocket and HA. The Y218N mutation propagated its effect by increasing the HB-HC interhelical angle, which in turn disrupted the packing around F198. Furthermore, a nonnative contact formed between E221 and S132 on the S1-HA loop, which offered a direct mechanism for disrupting the hydrophobic packing between the S1-HA loop and HC. While there were common misfolding features shared between Y218N and E196K, the differences in the orientation of HB and HC and the X-loop conformation might provide a structural basis for identifying different prion strains.

The folding pathway of the small α/βprotein GB1 has been extensively studied during the past two decades using both theoretical and experimental approaches. These studies provided a consensus view that the protein folds in a two-state manner. Here, we reassessed the folding of GB1, both by experiments and simulations, and detected the presence of an on-pathway intermediate. This intermediate has eluded earlier experimental characterization and is distinct from the collapsed state previously identified using ultrarapid mixing. Failure to identify the presence of an intermediate affects some of the conclusions that have been drawn for GB1, a popular model for protein folding studies.

Transmissible spongiform encephalopathies, or prion diseases, are caused by misfolding and aggregation of the prion protein PrP. Conversion from the normal cellular form (PrPC) or recombinant PrP (recPrP) to a misfolded form is pH-sensitive, in that misfolding and aggregation occur more readily at lower pH. To gain more insight into the influence of pH on the dynamics of PrP and its potential to misfold, we performed extensive molecular-dynamics simulations of the recombinant PrP protein (residues 90–230) in water at three different pH regimes: neutral (or cytoplasmic) pH (∼7.4), middle (or endosomal) pH (∼5), and low pH (<4). We present five different simulations of 50 ns each for each pH regime, amounting to a total of 750 ns of simulation time. A detailed analysis and comparison with experiment validate the simulations and lead to new insights into the mechanism of pH-induced misfolding. The mobility of the globular domain increases with decreasing pH, through displacement of the first helix and instability of the hydrophobic core. At middle pH, conversion to a misfolded (PrPSc-like) conformation is observed. The observed changes in conformation and stability are consistent with experimental data and thus provide a molecular basis for the initial steps in the misfolding process.

The ultrafast folding pathway of the engrailed homeodomain has been exceptionally well characterized by experiment and simulation. Helices II and III of the three-helix bundle protein form the native helix-turn-helix motif as an on-pathway intermediate within a few microseconds. The slow step is then the proper docking of the helices in ∼15 μs. However, there is still the unexplained puzzle of why helix docking is relatively slow, which is part of the more general question as to why rearrangements of intermediates occur slowly. To address this problem, we performed 46 all-atom molecular dynamics refolding simulations in explicit water, for a total of 15 μs of simulation time. The simulations started from an intermediate state structure that was generated in an unfolding simulation at 498 K and was then quenched to folding-permissive temperatures. The protein refolded successfully in only one of the 46 simulations, and in that case the refolding pathway mirrored the unfolding pathway at high temperature. In the 45 simulations in which the protein did not fully fold, nonnative salt bridges trapped the protein, which explains why the protein folds relatively slowly from the intermediate state.

The goal of the Dynameomics project is to perform, store, and analyze molecular dynamics simulations of representative proteins, of all known globular folds, in their native state and along their unfolding pathways. To analyze unfolding simulations, the location of the protein along the unfolding reaction coordinate (RXN) must be determined. Properties such as the fraction of native contacts and radius of gyration are often used; however, there is an issue regarding degeneracy with these properties, as native and nonnative species can overlap. Here, we used 15 physical properties of the protein to construct a multidimensional-embedded, one-dimensional RXN coordinate that faithfully captures the complex nature of unfolding. The unfolding RXN coordinates for 188 proteins (1534 simulations and 22.9 μs in explicit water) were calculated. Native, transition, intermediate, and denatured states were readily identified with the use of this RXN coordinate. A global native ensemble based on the native-state properties of the 188 proteins was created. This ensemble was shown to be effective for calculating RXN coordinates for folds outside the initial 188 targets. These RXN coordinates enable, high-throughput assignment of conformational states, which represents an important step in comparing protein properties across fold space as well as characterizing the unfolding of individual proteins.

The Dynameomics project aims to simulate a representative sample of all globular protein metafolds under both native and unfolding conditions. We have identified protein unfolding transition state (TS) ensembles from multiple molecular dynamics simulations of high-temperature unfolding in 183 structurally distinct proteins. These data can be used to study individual proteins and individual protein metafolds and to mine for TS structural features common across all proteins. Separating the TS structures into four different fold classes (all proteins, all-α, all-β, and mixed α/β and α +β) resulted in no significant difference in the overall protein properties. The residues with the most contacts in the native state lost the most contacts in the TS ensemble. On average, residues beginning in an α-helix maintained more structure in the TS ensemble than did residues starting in β-strands or any other conformation. The metafolds studied here represent 67% of all known protein structures, and this is, to our knowledge, the largest, most comprehensive study of the protein folding/unfolding TS ensemble to date. One might have expected broad distributions in the average global properties of the TS relative to the native state, indicating variability in the amount of structure present in the TS. Instead, the average global properties converged with low standard deviations across metafolds, suggesting that there are general rules governing the structure and properties of the TS.

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron degenerative disease, and the inherited form, familial ALS (fALS), has been linked to over 100 different point mutations scattered throughout the Cu-Zn superoxide dismutase protein (SOD1). The disease is likely due to a toxic gain of function caused by the misfolding, oligomerization, and eventual aggregation of mutant SOD1, but it is not yet understood how the structurally diverse mutations result in a common disease phenotype. The behavior of the apo-monomer fALS-associated mutant protein A4V was explored using molecular-dynamics simulations to elucidate characteristic structural changes to the protein that may allow the mutant form to improperly associate with other monomer subunits. Simulations showed that the mutant protein is less stable than the WT protein overall, with shifts in residue-residue contacts that lead to destabilization of the dimer and metal-binding sites, and stabilization of nonnative contacts that leads to a misfolded state. These findings provide a unifying explanation for disparate experimental observations, allow a better understanding of alterations of residue contacts that accompany loss of SOD1 structural integrity, and suggest sites where compensatory changes may stabilize the mutant structure.

Molecular dynamics simulations of aquaporin-1 embedded in a solvated lipid bilayer were carried out to investigate the mechanism of water permeation. The 2.2 Å resolution crystal structure of the bovine protein was used for five independent trajectories. During the equilibration and preparatory steps in which the protein was held fixed, water molecules inside the water channel adopted the same positions as observed in the crystal structure but they did not pass through the channel, suggesting that the dynamic motion of the protein is critical for water permeation. When the protein atoms were allowed to move, the side chains of the two asparagines in the two conserved Asn-Pro-Ala motifs near the center of the channel formed hydrogen bonds with water and helped water molecules move through the channel by actively aligning them for transport. The main-chain oxygen atoms, which were exposed to the pore surface in the crystal structure, also contributed to water transfer. Besides the constriction region observed in the crystal structure (Arg197, Phe58, His182, and Cys191), we found that His76 and Val155 act as a valve by dynamically blocking water permeation and helping control flow.

A properly identified transition state ensemble (TSE) in a molecular dynamics (MD) simulation can reveal a tremendous amount about how a protein folds and offer a point of comparison to experimentally derived ΦF values, which reflect the degree of structure in these transient states. In one such method of TSE identification, dubbed Pfold, MD simulations of individual protein structures taken from an unfolding trajectory are used to directly assess an input structure's probability of folding before unfolding, and Pfold is, by definition, 0.5 for the TSE. Other, less computationally intensive methods, such as multidimensional scaling (MDS) of the pairwise root mean-squared deviation (RMSD) matrix of the conformations sampled in a thermal unfolding trajectory, have also been used to identify the TSE. Identification of the TSE is made from the original MD simulation without the need to run further simulations. Here we present a Pfold-like study and describe methods for identification of the TSE through the derivation of a high fidelity, bounded, one-dimensional reaction coordinate for protein folding. These methods are applied to the engrailed homeodomain. The TSE identified by this approach is essentially identical to the TSE identified previously by MDS of the pairwise RMSD matrix. However, the cost of performing Pfold, or even our reduced Pfold-like calculations, is at least 36,000 times greater than the MDS method.

A novel secondary structure, the -sheet, was identified through molecular dynamics (MD) simulations of various proteins associated with amyloid diseases under amyloidogenic conditions. The structure was first predicted by Pauling and Corey, and it has been directly observed in crystal structures of “nonnatural peptides”. There are occurrences of -strands and -sheets in the Protein Data Bank, but they are rare. We propose that -sheet is formed during the conformational changes associated with amyloidosis and that it may represent the toxic conformer. Here, structural properties of the -sheet, background information, and experimental support for this novel structure are presented. Finally we speculate about the possible role of this conformation in disease.

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 β-turns and short distorted β-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. © 2002 Wiley Periodicals, Inc. Biopolymers 68: 121–129, 2003

Elastin is a cross-linked protein in the extracellular matrix that provides elasticity for many tissues. Its soluble precursor (tropoelastin) has two major types of alternating domains: (1) hydrophilic cross-linked domains rich in Lys and Ala and (2) hydrophobic domains (responsible for elasticity) rich in Val, Pro, and Gly, which often occur in repeats of VPGVG or VGGVG. Since native elastin is large and insoluble, many studies have focused on elastin-based peptides in an effort to elucidate its structure–function relationship. This review focuses on the molecular basis of elastin's conformational properties and associated elasticity. From both experimental and simulation approaches, elastin can be described as a two-phase model consisting of dynamic hydrophobic domains in water. The hydrophobic domain of elastin is best described as a compact amorphous structure with distorted β-strands, fluctuating turns, buried hydrophobic residues, and main-chain polar atoms that form hydrogen bonds with water. Water plays a critical role in determining elastin's conformational behavior, making elastin extremely dynamic in its relaxed state and providing an important source of elasticity.

As computer power increases, so too does the range of interesting biomolecular phenomena and properties that can be simulated. It is now possible to simulate complicated protein conformational changes at ambient or physiological temperatures. In this regard, we are attempting to map the conformational transitions of the normal, cellular prion protein (PrPC) to its infectious scrapie isoform (PrPSc), which causes neurodegenerative diseases in many mammals. These two forms have identical sequences and are conformational isomers, with heightened formation of β–sheet structure in the scrapie form. Conversion can be triggered by lowering the pH, but thus far it has been impossible to characterize the conformational change at high resolution using experimental methods. Therefore, to investigate the effect of acidic pH on PrP conformation, we have performed molecular–dynamics simulations of hamster, human and bovine forms of the prion protein in water at neutral and low pH. In all cases the core of the protein is well maintained at neutral pH. At low pH, however, the protein is more dynamic, and the sheet–like structure increases both by lengthening of the native β–sheet and by addition of a portion of the N–terminus to widen the sheet by another 2–3 strands.

Mutations in domain 2 (D2, residues 151–266) of the actin-binding protein gelsolin cause familial amyloidosis–Finnish type (FAF). These mutations, D187N or D187Y, lead to abnormal proteolysis of plasma gelsolin at residues 172–173 and a second hydrolysis at residue 243, resulting in an amyloidogenic fragment. Here we present the structure of human gelsolin D2 at 1.65 Å and find that Asp 187 is part of a Cd2+metal-binding site. Two Ca2+ions are required for a conformational transition of gelsolin to its active form. Differential scanning calorimetry (DSC) and molecular dynamics (MD) simulations suggest that the Cd2+-binding site in D2 is one of these two Ca2+-binding sites and is essential to the stability of D2. Mutation of Asp 187 to Asn disrupts Ca2+binding in D2, leading to instabilities upon Ca2+activation. These instabilities make the domain a target for aberrant proteolysis, thereby enacting the first step in the cascade leading to FAF.