The V108M mutation decreases the structural stability of catechol O-methyltransferase.

The human gene for catechol O-methyltransferase has a common single-nucleotide polymorphism that results in substitution of methionine (M) for valine (V) 108 in the soluble form of the enzyme (s-COMT). 108M s-COMT loses enzymatic activity more rapidly than 108V s-COMT at physiological temperature, and the 108M allele has been associated with increased risk of breast cancer and several neuropsychiatric disorders. We used circular dichroism (CD), dynamic light scattering, and fluorescence spectroscopy to examine how the 108V/M polymorphism affects the stability of the purified, recombinant protein to heat and guanidine hydrochloride (GuHCl). COMT contains two tryptophan residues, W143 and W38Y, which are located in loops that border the S-adenosylmethionine (SAM) and catechol binding sites. We therefore also studied the single-tryptophan mutants W38Y and W143Y in order to dissect the contributions of the individual tryptophans to the fluorescence signals. The 108V and 108M proteins differed in the stability of both the tertiary structure surrounding the active site, as probed by the fluorescence yields and emission spectra, and their global secondary structure as reflected by CD. With either probe, the midpoint of the thermal transition of 108M s-COMT was 5 to 7 degrees C lower than that of 108V s-COMT, and the free energy of unfolding at 25 degrees C was smaller by about 0.4 kcal/mol. 108M s-COMT also was more prone to aggregation or partial unfolding to a form with an increased radius of hydration at 37 degrees C. The co-substrate SAM stabilized the secondary structure of both 108V and 108M s-COMT. W143 dominates the tryptophan fluorescence of the folded protein and accounts for most of the decrease in fluorescence that accompanies unfolding by GuHCl. While replacing either tryptophan by tyrosine was mildly destabilizing, the lower stability of the 108M variant was retained in all cases.

Selective neuronal targeting in prion disease.

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

Long timescale simulations.

Computers are becoming increasingly fast, making it possible to perform simulations of macromolecules on timescales that were previously inaccessible. Questions have arisen concerning how well we are keeping up with computer power and the state of the art with respect to long molecular dynamics simulations in solvent. More importantly, however, simulations of macromolecules are performed to aid the understanding of biochemical phenomena. So, what are we learning from longer simulations and are they providing reliable insight into protein dynamics, conformational behavior and function?

The prion folding problem.

Prion diseases are neurodegenerative disorders in which dramatic conformational change in the structure of the prion protein is the fundamental event. This structural transition involves the loss of substantial alpha-helical content and the acquisition of beta-sheet structure. A convergence of recent biological and structural studies argues that the mechanism underlying the prion diseases is truly unprecedented.

Characterization of the unfolding pathway of the cell-cycle protein p13suc1 by molecular dynamics simulations: implications for domain swapping.

BACKGROUND: The p13suc1 gene product is a member of the cks (cyclin-dependent protein kinase subunit) protein family and has been implicated in regulation of the cell cycle. Various crystal structures of suc1 are available, including a globular, monomeric form and a beta-strand exchanged dimer. It has been suggested that conversions between these forms, and perhaps others, may be important in the regulation of the cell cycle. RESULTS: We have undertaken molecular dynamics simulations of protein unfolding to investigate the conformational properties of suc1. Unfolding transition states were identified for each of four simulations. These states contain some native secondary structure, primarily helix alpha1 and the core of the beta sheet. The hydrophobic core is loosely packed. Further unfolding leads to an intermediate state that is slightly more expanded than the transition state, but with considerably fewer nonlocal, tertiary packing contacts and less secondary structure. The helices are fluctuating but partially formed in the denatured state and beta2 and beta4 remain associated. CONCLUSIONS: It appears that suc1 folds by a nucleation-condensation mechanism, similar to that observed for two-state folding proteins. However, suc1 forms an intermediate during unfolding and contains considerable residual structure in the denatured state. The stability of the beta2-beta4 residual structure is surprising, because beta4 is the strand involved in domain swapping. This stability suggests that the domain-swapping event, if physiologically relevant, may require the assistance of additional factors in vivo or occur early in the folding process.

Protein unfolding pathways explored through molecular dynamics simulations.

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.

Molecular dynamics simulations of helix denaturation.

An understanding of the structural transitions that an alpha-helix undergoes will help to elucidate such motions in proteins and their role in protein folding. We present the results of molecular dynamics simulations to investigate these transitions in a short polyalanine peptide (13 residues) both in vacuo and in the presence of solvent. The denaturation of this peptide was monitored as a function of temperature (ranging from 5 to 200 degrees C). In vacuo, the helical state predominated at all temperatures, whereas in solution the helix melted with increasing temperature. The peptide was predominantly helical at low temperature in solution, while at intermediate temperatures the peptide spent the bulk of the time fluctuating between different conformations with intermediate amounts of helix, e.g. not completely helical nor entirely non-helical. Many of these conformations consisted of short helical segments with intervening non-helical residues. At high temperature the peptide unfolded and adopted various collapsed unstructured states. The intrahelical hydrogen bonds that break at high temperature were not fully compensated by hydrogen bonds with water molecules in the partially unfolded forms of the peptide. Increases in temperature disrupted both the helical structure and the peptide-water interactions. Water played a major but indirect role in facilitating unfolding, as opposed to specifically competing for the intrapeptide hydrogen bonds. The implications of our results to protein folding are discussed.

Molecular dynamics simulation of the unfolding of barnase: characterization of the major intermediate.

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 core1 and core3 being slightly stronger than core2. Helix alpha 1 is substantially formed, with the center being stronger than the termini. The first turn of alpha 2 is lost and alpha 3 is unfolded. The center of the beta-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 S and the experimentally measured phi values, which are based on energetics, except for three residues. These residues are polar and non-conservative mutations were made to obtain phi values, which can complicate structural interpretations. These residues make strong side-chain interactions in I1, but the backbone structure is disrupted, leading to low S values. Thus, this discordance highlights possible limitations in both the phi value and S value analyses: strong polar interactions in the intermediate may give rise to high phi 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.

Identification and characterization of the unfolding transition state of chymotrypsin inhibitor 2 by molecular dynamics simulations.

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 > or = 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 coefficient R = 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.

Molecular dynamics simulations of protein unfolding and limited refolding: characterization of partially unfolded states of ubiquitin in 60% methanol and in water.

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.

Simulations of the structural and dynamical properties of denatured proteins: the "molten coil" state of bovine pancreatic trypsin inhibitor.

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 beta-structure or the opposite. To determine whether the protein preferentially sampled particular conformational substates in the denatured state, pairwise Calpha 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.

Non-native interactions in protein folding intermediates: molecular dynamics simulations of hen lysozyme.

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 alpha-domain and a structured beta-domain, which is inconsistent with experiment. In contrast, the beta-domain unfolded first in the two simulations presented here leaving a structured alpha-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.

Analysis methods for comparison of multiple molecular dynamics trajectories: applications to protein unfolding pathways and denatured ensembles.

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 via expansion 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 via different 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".

The molecular basis for the inverse temperature transition of elastin.

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 degrees C, for a total of 80 ns. Beginning from an idealized beta-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 beta-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 beta II 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 beta-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 approximately 40 % local beta-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.

Mapping the interactions present in the transition state for unfolding/folding of FKBP12.

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 alpha-spectrin. For FKBP12, the central three strands of the beta-sheet, beta-strand 2, beta-strand 4 and beta-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 beta-strand, makes approximately 60 % of its native interactions. The outer beta-strands and the ends of the central beta-strands are formed to a lesser degree. The short alpha-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 beta-strands 2, 4 and 5, and the C terminus of the alpha-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 Bronsted analysis confirming that the transition state is an ensemble of states close in structure.

Structure of the transition state for folding of a protein derived from experiment and simulation.

Independent experimental and theoretical studies of the unfolding of barley chymotrypsin inhibitor 2 (CI2) are compared in an attempt to derive plausible three-dimensional structural models of the transition states. A very simple structure index is calculated along the sequence for the molecular dynamics-generated transition state models to facilitate comparison with the phi F values. The two are in good agreement overall (correlation coefficient = 0.87), which suggests that the theoretical models should provide a structural framework for interpretation of the phi F values. Both experiment and simulation indicate that the transition state is a distorted form of the native state in which the alpha-helix is weakened but partially intact and the beta-sheet is quite disrupted. As inferred from the phi f values and observed directly in the simulations, the unfolding of CI2 is cooperative and there is a "folding core" comprising a patch on the alpha-helix and a portion of the beta-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 phi 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 phi F values 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.

Towards a complete description of the structural and dynamic properties of the denatured state of barnase and the role of residual structure in folding.

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 beta(3-4) hairpin contained a particularly high degree of such side-chain interactions but it lacked beta-structure in two of the three denatured ensembles: beta(3-4) was the only portion of the beta-structure to contain significant residual structure in the denatured state. The two principal alpha-helices (alpha1 and alpha2) 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 alpha1 and alpha2, as well as fluctuating tertiary contacts in the beta(3-4) region, and between alpha1 and beta(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 beta(3-4) and alpha1 assisted in the refolding of alpha1. This contact-assisted helix formation was confirmed in fragment simulations of beta(3-4) and alpha1 alone and complexed, and, as such, alpha1 and beta(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 beta(3-4) and alpha1 are dynamic in the denatured state but when they collide and make enough contacts, they provide a loose structural scaffold onto which further beta-strands pack. The beta-structure condenses about beta(3-4), while alpha1 aids in stabilizing beta(3-4) and maintaining its orientation. The resulting beta-structure is relatively planar and loose in the major intermediate. Further packing ensues, and as a result the beta-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.

Theoretical studies of sequence effects on the conformational properties of a fragment of the prion protein: implications for scrapie formation.

BACKGROUND: Prion diseases are neurodegenerative disorders that appear to be due to a conformational change, involving the conversion of alpha-helices in the normal, cellular isoform of the prion protein (PrPC) to beta-structure in the infectious scrapie form (PrPSc). One form of Gerstmann-Sträussler-Scheinker syndrome (GSS), an inherited prion disease, is caused by mutation of Ala117 of PrPC to Val. We therefore set out to evaluate the effects of this mutation on the stability of the PrPC form. RESULTS: We have performed molecular dynamics simulations of a portion of the PrPC sequence (residues 109-122, termed H1) that is proposed to figure prominently in the conversion of PrPC to PrPSc. In particular, beginning with H1 in the alpha-helical state, the conformational consequences of sequence changes at position 117 were investigated for six hydrophobic mutations. Of these, only the Val mutation was helix-destabilizing. Portions of this mutant peptide adopted and retained an extended conformation during a 2 ns simulation of the peptide in water. CONCLUSIONS: The conformational transitions and structures observed in the simulation of the mutant peptide with Val at position 117 provide insight into the possible early steps in the conversion of PrPC to PrPSc.

Structure-function aspects of prion proteins.

Prions diseases are fatal neurodegenerative disorders resulting from conformational changes in the prion protein from the normal cellular form, PrPC, to the infectious scrapie isoform, PrPSc. High resolution structures for PrPC are now available, and biochemical investigations are shedding light on the nature and determinants of the conformational transition. Together, these studies are beginning to provide a framework to describe structure-function relationships of the prion protein.

Molecular dynamics simulations of hydrophobic collapse of ubiquitin.

Nine nonnative conformations of ubiquitin, generated during two different thermal denaturation trajectories, were simulated under nearly native conditions (62 degrees C). The simulations included all protein and solvent atoms explicitly, and simulation times ranged from 1-2.4 ns. The starting structures had alpha-carbon root-mean-square deviations (RMSDs) from the crystal structure of 4-12 A and radii of gyration as high as 1.3 times that of the native state. In all but one case, the protein collapsed when the temperature was lowered and sampled conformations as compact as those reached in a control simulation beginning from the crystal structure. In contrast, the protein did not collapse when simulated in a 60% methanol:water mixture. The behavior of the protein depended on the starting structure: during simulation of the most native-like starting structures (<5 A RMSD to the crystal structure) the RMSD decreased, the number of native hydrogen bonds increased, and the secondary and tertiary structure increased. Intermediate starting structures (5-10 A RMSD) collapsed to the radius of gyration of the control simulation, hydrophobic residues were preferentially buried, and the protein acquired some native contacts. However, the protein did not refold. The least native starting structures (10-12 A RMSD) did not collapse as completely as the more native-like structures; instead, they experienced large fluctuations in radius of gyration and went through cycles of expansion and collapse, with improved burial of hydrophobic residues in successive collapsed states.