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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.

Atomic force microscopy (AFM) experiments have provided intriguing insights into the mechanical unfolding of proteins such as titin I27 from muscle, but will the same be possible for proteins that are not physiologically required to resist force? We report the results of AFM experiments on the forced unfolding of barnase in a chimeric construct with I27. Both modules are independently folded and stable in this construct and have the same thermodynamic and kinetic properties as the isolated proteins. I27 can be identified in the AFM traces based on its previous characterization, and distinct, irregular low-force peaks are observed for barnase. Molecular dynamics simulations of barnase unfolding also show that it unfolds at lower forces than proteins with mechanical function. The unfolding pathway involves the unraveling of the protein from the termini, with much more native-like secondary and tertiary structure being retained in the transition state than is observed in simulations of thermal unfolding or experimentally, using chemical denaturant. Our results suggest that proteins that are not selected for tensile strength may not resist force in the same way as those that are, and that proteins with similar unfolding rates in solution need not have comparable unfolding properties under force.

Experimental and simulation studies can complement each other nicely in the area of protein folding. Experiment reports on the average properties of a large ensemble (approx. 10(17)-10(19) molecules), typically over time. Molecular dynamics simulations, on the other hand, provide detailed information for a single molecule, a component of the ensemble. By combining these approaches we can obtain not only a more complete picture of folding, but we can also take advantage of the strengths of different methods. For example, experiment cannot provide molecular structures. Molecular dynamics simulations can provide such information, but the simulations are meaningless without a linked experiment. Thus, the interrelated nature of simulation in assessing experimental assumptions and in providing structures to augment energetic descriptions, and experiment in judging whether the simulations are reasonable, provides more confidence in the resulting information about folding. This combination yields tested and testable molecular models of states that evade characterization by conventional methods. Therefore, we have explored the combined use of these methods to map folding/unfolding pathways at atomic resolution, in collaboration with Alan Fersht. Here we focus on chymotrypsin inhibitor 2, a small single-domain, two-state folding protein.

RetGC-1, a member of the membrane guanylyl cyclase family of proteins, is regulated in photoreceptor cells by a Ca2+-binding protein known as GCAP-1. Proper regulation of RetGC-1 is essential in photoreceptor cells for normal light adaptation and recovery to the dark state. In this study we show that cGMP synthesis by RetGC-1 requires dimerization, because critical functions in the catalytic site must be provided by each of the two polypeptide chains of the dimer. We also show that an intact α-helical coiled-coil structure is required to provide dimerization strength for the catalytic domain of RetGC-1. However, the dimerization strength of this domain must be precisely optimized for proper regulation by GCAP-1. We found that Arg838within the dimerization domain establishes the Ca2+sensitivity of RetGC-1 by determining the strength of the coiled-coil interaction. Arg838substitutions dominantly enhance cGMP synthesis even at the highest Ca2+concentrations that occur in normal dark-adapted photoreceptor cells. Molecular dynamics simulations suggest that Arg838substitutions disrupt a small network of salt bridges to allow an abnormal extension of coiled-coil structure. Substitutions at Arg838were first identified by linkage to the retinal degenerative disease, autosomal dominant cone rod dystrophy (adCORD). Consistent with the characteristics of this disease, the Arg838-substituted RetGC-1 mutants exhibit a dominant biochemical phenotype. We propose that accelerated cGMP synthesis in humans with adCORD is the primary cause of cone-rod degeneration.

The folding pathway of FKBP12, a 107 residue α/β protein, has been characterised in detail using a combination of experimental and computational techniques. FKBP12 follows a two-state model of folding in which only the denatured and native states are significantly populated; no intermediate states are detected. The refolding rate constant in water is 4 s-1at 25 °C. Two different experimental strategies were employed for studying the transition state for folding. In the first case, a non-mutagenic approach was used and the unfolding and refolding of the wild-type protein measured as a function of experimental conditions such as temperature, denaturant, ligand and trifluoroethanol (TFE) concentration. These data suggest a compact transition state relative to the unfolded state with some 70% of the surface area buried. The ligand-binding site, whichis mainly formed by two long loops, is largely unstructured in the transition state. TFE experiments suggest that the α-helix may be formed in the transition state. The second experimental approach involved using protein engineering techniques with φ-value analysis. Residue-specific information on the structure and energetics of the transition state can be obtained by this method. 34 mutations were made at sites throughout the protein to probe the extent of secondary and tertiary structure in the transition state. In contrast to some other proteins of this size, no element of structure is fully formed in the transition state, instead, the transition state is similar to that found for smaller, single-domain proteins, such as chymotrypsin inhibitor 2 and the SH3 domainfrom α-spectrin. For FKBP12, the central three strands of the β-sheet (2, 4 and 5), comprise the most structured region of the transition state. In particular Val 101, which is one of the most highly buried residues and located in the middle of the central β-strand,makes approximately 60% of its native interactions. The outer β-strands, and the ends of the central β-strands are formed to a lesser degree. The short α-helix is largely unstructured in the transition state as are the loops. The data are consistent with a nucleation-condensation model of folding, the nucleus of which is formed by side chains within β-strands 2, 4 and 5 and the C-terminus of the α-helix. These residues are distant in the primary sequence, demonstrating the importance of tertiary interactions in the transition state. High-temperature molecular dynamic simulations on the unfoldingpathway of FKBP12 are in good agreement with the experimental results.

Elastin undergoes an “inverse temperature transition” such that it becomes more ordered as the temperature increases. To investigate the molecular basis for this behavior, molecular dynamics simulations were conducted above and below the transition temperature. Simulations of a 90-residue elastin peptide, (VPGVG)18, with explicit water molecules were performed at seven different temperatures between 7 and 42 °C, for a total of 80 ns. Beginning from an idealized β-spiral structure, hydrophobic collapse was observed over a narrow temperature range in the simulations. Moreover, simulations above and below elastin’s transition temperature indicate that elastin has more turns and distorted β-structure at higher temperatures. Water was critical to the inverse temperature transition and elastin-associated water molecules can be divided into three categories: those closely associated with βII turns; those that form hydrogen bonds with the main-chain groups; and those hydrating the hydrophobic side-chains. Water-swollen, monomeric elastin above the transition temperature is best described as a compact amorphous structure with distorted β-strands, fluctuating turns, buried hydrophobic residues, and main-chain polar atoms that participate in hydrogen bonds with water. Below the transition temperature, elastin is expanded with ∼40 % local β-spiral structure. Overall the simulations are in agreement with experiment and therefore appear to provide an atomic-level description of the conformational properties of elastin monomers and the basis for their elastomeric properties.

The detailed characterization of denatured proteins remains elusive due to their mobility and conformational heterogeneity. NMR studies are beginning to provide clues regarding residual structure in the denatured state but the resulting data are too sparse to be transformed into molecular models using conventional techniques. Molecular dynamics simulations can complement NMR by providing detailed structural information for components of the denatured ensemble. Here, we describe three independent 4 ns high-temperature molecular dynamics simulations of barnase in water. The simulated denatured state was conformationally heterogeneous with respect to the conformations populated both within a single simulation and between simulations. Nonetheless, there were some persistent interactions that occurred to varying degrees in all simulations and primarily involved the formation of fluid hydrophobic clusters with participating residues changing over time. The region of the β(3–4) hairpin contained a particularly high degree of such side-chain interactions but it lacked β-structure in two of the three denatured ensembles: β(3–4) was the only portion of the β-structure to contain significant residual structure in the denatured state. The two principal α-helices (α1 and α2) adopted dynamic helical structure. In addition, there were persistent contacts that pinched off core 2 from the body of the protein. The rest of the protein was unstructured, aside from transient and mostly local side-chain interactions. Overall, the simulated denatured state contains residual structure in the form of dynamic, fluctuating secondary structure in α1 and α2, as well as fluctuating tertiary contacts in the β(3–4) region, and between α1 and β(3–4), in agreement with previous NMR studies. Here, we also show that these regions containing residual structure display impaired mobility by both molecular dynamics and NMR relaxation experiments. The residual structure was important in decreasing the conformational states available to the chain and in repairing disrupted regions. For example, tertiary contacts between β(3–4) and α1 assisted in the refolding of α1. This contact-assisted helix formation was confirmed in fragment simulations of β(3–4) and α1 alone and complexed, and, as such, α1 and β(3–4) appear to be folding initiation sites. The role of these sites in folding was investigated by working backwards and considering the simulation in reverse, noting that earlier time-points from the simulations provide models of the major intermediate and transition states in quantitative agreement with data from both unfolding and refolding experiments. Both β(3–4) and α1 are dynamic in the denatured state but when they collide and make enough contacts, they provide a loose structural scaffold onto which further β-strands pack. The β-structure condenses about β(3–4), while α1 aids in stabilizing β(3–4) and maintaining its orientation. The resulting β-structure is relatively planar and loose in the major intermediate. Further packing ensues, and as a result the β-sheet twists, leading to the major transition state. The structure is still expanded and loops are not well formed at this point. Fine-tuning of the packing interactions and the final condensation of the structure then occurs to yield the native state.

The structure of the transition state for folding/unfolding of the immunophilin FKBP12 has been characterised using a combination of protein engineering techniques, unfolding kinetics, and molecular dynamics simulations. A total of 34 mutations were made at sites throughout the protein to probe the extent of secondary and tertiary structure in the transition state. The transition state for folding is compact compared with the unfolded state, with an approximately 30 % increase in the native solvent-accessible surface area. All of the interactions are substantially weaker in the transition state, as probed by both experiment and molecular dynamics simulations. In contrast to some other proteins of this size, no element of structure is fully formed in the transition state; instead, the transition state is similar to that found for smaller, single-domain proteins, such as chymotrypsin inhibitor 2 and the SH3 domain from α-spectrin. For FKBP12, the central three strands of the β-sheet, β-strand 2, β-strand 4 and β-strand 5, comprise the most structured region of the transition state. In particular Val101, which is one of the most highly buried residues and located in the middle of the central β-strand, makes approximately 60 % of its native interactions. The outer β-strands and the ends of the central β-strands are formed to a lesser degree. The short α-helix is largely unstructured in the transition state, as are the loops. The data are consistent with a nucleation-condensation model of folding, the nucleus of which is formed by side-chains within β-strands 2, 4 and 5, and the C terminus of the α-helix. The precise residues involved in the nucleus differ in the two simulated transition state ensembles, but the interacting regions of the protein are conserved. These residues are distant in the primary sequence, demonstrating the importance of tertiary interactions in the transition state. The two independently derived transition state ensembles are structurally similar, which is consistent with a Brønsted analysis confirming that the transition state is an ensemble of states close in structure. Copyright 1999 Academic Press

In molecular dynamics simulations of protein unfolding, the pathway of one protein molecule is studied at a time. In contrast, experimental denaturation studies sample from large ensembles of molecules passing from the native to unfolded state. If reasonable comparisons with experiment are to be made, then the generality of the simulations needs to be confirmed by performing multiple unfolding simulations. Given that protein unfolding trajectories are very complicated functions of the proteins and the environment, comparing different trajectories, even under the same conditions, is not straightforward. Several methods are presented here that attempt to accomplish this task at different levels of complexity. The simpler methods are geometry based and make use of the root-mean-squared deviations between structures, while the more complicated methods are based on the time variation of the various properties of the system during the unfolding process. These methods are applied to multiple simulations of three different proteins, bovine pancreatic trypsin inhibitor, chymotrypsin inhibitor 2, and barnase. In general, for these three proteins protein unfolding proceeded viaexpansion of the core and fraying of secondary structure to yield the major transition state. Once past the transition state, the trajectories for a given protein diverged as the protein lost further secondary and tertiary structure by a variety of mechanisms. Although the unfolding pathways diverged, similar conformations were populated in the denatured state even when the unfolding occurred viadifferent pathways. The multitude of different pathways leading to the denatured state agrees with the funnel description of protein folding. Although the pathways differed in conformational space, the physical properties of the conformations were often similar, highlighting the danger of assuming that similar observed properties imply similar conformations. In fact, there may be many different “conformational pathways” of unfolding that fit within a preferred “property space pathway”.

Molecular dynamics simulations of protein denaturation can complement and extend experimental studies of protein folding by providing atomic-level structural information about conformational transitions and any conformational states along the unfolding pathway. Previous unfolding simulations of hen egg-white lysozyme have resulted in intermediate structures with an unfolded α-domain and a structured β-domain, which is inconsistent with experiment. In contrast, the β-domain unfolded first in the two simulations presented here leaving a structured α-domain. Following this, intermediate states were identified that differ with respect to the packing of the helices and the elements of non-native structure adopted. The non-native structure is critical for explaining many of the experimental observations. Overall, the pooled ensemble of these intermediates is in agreement with the experimental data for the major kinetic intermediate, suggesting that the kinetic intermediate may be made up of distinct, but rapidly interconverting, partially folded conformations distinguished primarily by differences in helix packing.

Independent experimental and theoretical studies of the unfolding of barley chymotrypsin inhibitor 2 (C/2) are compared in an attempt to derive plausible three-dimensional structural models of the transition state. A very simple structure index is calculated along the sequence for the molecular dynamics-generated transition state models to facilitate comparison with the_Fvalues. The two are in good agreement overall (correlation coefficient=0.87), which suggests that the theoreticalmodels should provide a structural framework for interpretation of the_Fvalues. Both experiment and simulation indicate that the transition state is a distorted form of the native state in which the α-helix is weakened but partially intact and the β-sheet is quite disrupted. As inferred from the_Fvalues and observed directly in the simulations, the unfolding of CI2 is cooperative and there is a “folding core” comprising a patch on the α-helix and a portion of the β-sheet, nucleated by interactions between Ala16, Ile49 and other neighbouring residues. The protein becomes less structured radiating away from this core. Overall the data indicate that CI2 folds by a nucleation-collapse mechanism. In the absence of experimental information, we have little confidence that the molecular dynamics simulations are correct, especially when only one or a few simulations are performed. On the other hand, even though the experimentally derived values may reflect the extent of overall structure formation, they do not provide an actual atomic-resolution three-dimensional structure of the transition state. By combining the two approaches, however, we have a framework for interpreting_Fvalues and can hopefully arrive at a more trustworthy model of the transition state. The process is in some ways similar to the combination of molecular dynamics and NMR data to solve the tertiary structure of proteins.

Extensive experimental data are available on the native, partially and fully unfolded states of ubiquitin. Two and three-dimensional NMR experiments of a partially unfolded form of the protein in 60% methanol indicate that approximately one-half of the molecule contains disrupted but native-like structure while the other half is unstructured and/or contains non-native structure. In contrast, the interpretation of hydrogen-exchange data have led to the conclusion that this state is native-like. Thus, there are discrepancies between the experimental studies, or interpretations based on the data. We compare the results of molecular dynamics simulations, under varying conditions, with the experimental results. The simulations extend past 0.5 ns and include explicit solvent molecules: either pure water or 60% methanol. To begin with, ubiquitin was thermally denatured in water (at 498 K). Two particular structures, or “aliquots”, during the unfolding process were selected for further study (60 and 198 ps). These structures were then simulated separately in water and 60% methanol at a lower and experimentally meaningful temperature (335 K). The conformations generated from the structure extracted later in the simulation contained significant amounts of non-native structure in the presence of methanol while satisfying both the NMR and hydrogen exchange data. In fact, clearly non-native regions of the structure yielded the desired protection from hydrogen exchange. In contrast, an earlier, more native-like, intermediate did not do as well at predicting the hydrogen-exchange behavior and was inconsistent with the NMR data. These data suggest that the results and interpretations using the different experimental techniques can be reconciled by a single state. This finding also brings into question the practice of interpreting protection to hydrogen exchange in terms of native secondary and tertiary structure, especially when one has weak patterns and low protection factors. When the partially unfolded states were placed in pure water, the protein collapsed and began to refold. Therefore, the desired solvent-dependent properties were observed: the partially unfolded conformations with increased exposure of hydrophobic residues remained expanded in methanol but collapsed in water as the non-polar groups minimized their exposure to solvent.

The folding/unfolding pathway of barnase has been studied extensively using the protein engineering method, which has provided indirect structural information for the transition state and the major folding intermediate. To further characterize the structural properties of the intermediate, we have simulated the thermal denaturation of barnase beginning from the average NMR structure. Our results indicate that there are at least two intermediates on the unfolding pathway. The three hydrophobic cores are partially formed in the major intermediate (I1), with core1and core3being slightly stronger than core2. Helix α1is substantially formed, with the center being stronger than the termini. The first turn of α2is lost and α3is unfolded. The center of the β-sheet is substantially formed, but the edges are disrupted. These structural characteristics are in good qualitative agreement with the experimental data. For semi-quantitative comparison with experimental data, the extent of native structure of individual residues is characterized by a structure index, S, that reflects both secondary and tertiary structure. There is good agreement between Sand the experimentally measured Φ values, which are based on energetics, except for three residues. These residues are polar and non-conservative mutations were made to obtain Φ values, which can complicate structural interpretations. These residues make strong side-chain interactions in I1, but the backbone structure is disrupted, leading to low Svalues. Thus, this discordance highlights possible limitations in both the Φ value and Svalue analyses: strong polar interactions in the intermediate may give rise to high Φ values that are not reflective of structure per se; however, due to sampling limitations, any one simulation is not expected to capture all of the features of the true conformational ensemble. In any case, these simulations provide an experimentally testable, atomic-level structural model for the major folding intermediate of barnase, as well as the detailed pathway from the native to the intermediate state.

A new conformational search method, molecular dynamics–minimization (MDM), is proposed, which combines a molecular dynamics sampling strategy with energy minimizations in the search for low‐energy molecular structures. This new method is applied to the search for low energy configurations of clusters of coulombic charges on a unit sphere, Lennard–Jones clusters, and water clusters. The MDM method is shown to be efficient in finding the lowest energy configurations of these clusters. A closer comparison of MDM with alternative conformational search methods on Lennard–Jones clusters shows that, although MDM is not as efficient as the Monte Carlo–minimization method in locating the global energy minima, it is more efficient than the diffusion equation method and the method of minimization from randomly generated structures. Given the versatility of the molecular dynamics sampling strategy in comparison to Monte Carlo in treating molecular complexes or molecules in explicit solution, one anticipates that the MDM method could be profitably applied to conformational search problems where the number of degrees of freedom is much greater. © 1998 John Wiley & Sons, Inc. J Comput Chem 19: 60–70, 1998

Temperature-induced unfolding of chymotrypsin inhibitor 2 (CI2) in water has been investigated using molecular dynamics simulations. One simulation (2.2 ns) has been analyzed in detail and three additional simulations (each≥1 ns) were performed to check the generality of the results. Concurrent loss of secondary and tertiary structure during unfolding was observed in all the simulations. For each simulation, the major transition state of unfolding was identified based on conformational analysis of protein structures along the unfolding trajectory. The transition state has a considerably weakened hydrophobic core and disrupted secondary structure. Nevertheless, the overall structure of the transition state is closer to the native state than to the unfolded state. The disruption of the hydrophobic core appears to be rate limiting. However, other energy barriers have to be overcome before reaching the major transition state. A method is described to quantitatively compare the structure of the simulated transition state with that characterized by protein engineering experiments. Good agreement with the experimental data is obtained for all four transition state models (the correlation coefficientR=0.80 to 0.93) and the average over all four models gives the best correlation (R=0.94). These simulations provide the first comprehensive atomic-level view of what the unfolding transition state of C12 may look like.

The pattern of scrapie prion protein (PrPSc) accumulation in the brain is different for each prion strain. We tested whether the PrPScdeposition pattern is influenced by the Asn-linked oligosaccharides of PrPCin transgenic mice. Deletion of the first oligosaccharide altered PrPCtrafficking and prevented infection with two prion strains. Deletion of the second did not alter PrPCtrafficking, permitted infection with one prion strain, and had a profound effect on the PrPScdeposition pattern. Our data raise the possibility that glycosylation can modify the conformation of PrPC. Glycosylation could affect the affinity of PrPCfor a particular conformer of PrPSc, thereby determining the rate of nascent PrPScformation and the specific patterns of PrPScdeposition.

The dynamic nature of denatured, unfolded proteins makes it difficult to characterize their structures experimentally. To complement experiment and to obtain more detailed information about the structure and dynamic behavior of the denatured state, we have performed eleven 2.5 ns molecular dynamics simulations of reduced bovine pancreatic trypsin inhibitor (BPTI) at high temperature in water and a control simulation at 298 K, for a total of 30 ns of simulation time. In a neutral pH environment (acidic residues ionized), the unfolded protein structures were compact with an average radius of gyration 9% greater than the native state. The compact conformations resulted from the transient formation of non-native hydrophobic clusters, turns and salt bridges. However, when the acidic residues were protonated, the protein periodically expanded to a radius of gyration of 18 to 20 A˚. The early steps in unfolding were similar in the different simulations until passing through the major transition state of unfolding. Afterwards, unfolding proceeded through one of two general pathways with respect to secondary structure: loss of the C-terminal helix followed by loss of β-structure or the opposite. To determine whether the protein preferentially sampled particular conformational substates in the denatured state, pairwise Cα root-mean-square deviations were measured between all structures, but similar structures were found between only two trajectories. Yet, similar composite properties (secondary structure content, side-chain and water contacts, solvent accessible surface area, etc.) were observed for the structures that unfolded through different pathways. Somewhat surprisingly, the unfolded structures are in agreement with both past experiments suggesting that reduced BPTI is a random coil and more recent experiments providing evidence for non-random structure, demonstrating how ensembles of fluctuating structures can give rise to experimental observables that are seemingly at odds.

Herein we describe the results of molecular dynamics simulations of the bovine pancreatic trypsin inhibitor (BPTI) in solution at a variety of temperatures both with and without disulfide bonds. The reduced form of the protein unfolded at high temperature to an ensemble of conformations with all the properties of the molten globule state. In this account we outline the structural details of the actual unfolding process between the native and molten globule states. The first steps of unfolding involved expansion of the protein, which disrupted packing interactions. The solvent-accessible surface area also quickly increased. The unfolding was localized mostly to the turn and loop regions of the molecule, while leaving the secondary structure intact. Then, there was more gradual unfolding of the secondary structure and non-native turns became prevalent. This same trajectory was continued and more drastic unfolding occurred that resulted in a relatively compact state devoid of stable secondary structure. Copyright 1993, 1999 Academic Press

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

We recently found that deletion of the Asn-linked carbohydrate (CHO) at residue 197 of Syrian hamster (SHa) PrPc while retaining the CHO at Asn 181 has a profound effect on which population of neurons are targeted for conversion of SHaPrPC to SHaPrPSc in transgenic (Tg) mice inoculated with scrapie prions. We hypothesized that selective targeting of neuronal populations is determined by cell-specific differences in the affinity of an infecting PrPSc (prion) for PrPC and that the affinity might be modulated by nerve cell-specific differences in PrPC glycosylation. Here we tested this hypothesis by assessing whether or not each brain region in Syrian hamsters synthesizes different PrPC glycoforms, as inferred from 2D-gel electrophoresis. Reproducible differences in the number and isoelectric point of PrPC charge isomers were found as a function of brain region. The results of this study support the hypothesis that the PrPSc accumulation and the vacuolation pattern phenotypes in the brain are governed by neuron-specific differences in PrPc glycoforms.