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Type 2 diabetes (T2D) results from insulin secretory dysfunction arising in part from the loss of pancreatic islet β‐cells. Several factors contribute to β‐cell loss, including islet amyloid formation, which is observed in over 90% of individuals with T2D. The amyloid is comprised of human islet amyloid polypeptide (hIAPP). Here we provide evidence that early in aggregation, hIAPP forms toxic oligomers prior to formation of amyloid fibrils. The toxic oligomers contain α‐sheet secondary structure, a nonstandard secondary structure associated with toxic oligomers in other amyloid diseases. De novo, synthetic α‐sheet compounds designed to be nontoxic and complementary to the α‐sheet structure in the toxic oligomers inhibit hIAPP aggregation and neutralize oligomer‐mediated cytotoxicity in cell‐based assays. In vivo administration of an α‐sheet design to mice for 4 weeks revealed no evidence of toxicity nor did it elicit an immune response. Furthermore, the α‐sheet designs reduced endogenous islet amyloid formation and mitigation of amyloid‐associated β‐cell loss in cultured islets isolated from an hIAPP transgenic mouse model of islet amyloidosis. Characterization of the involvement of α‐sheet in early aggregation of hIAPP and oligomer toxicity contributes to elucidation of the molecular mechanisms underlying amyloid‐associated β‐cell loss. [ABSTRACT FROM AUTHOR]

Proteins with similar structures are generally assumed to arise from similar sequences. However, there are more cases than not where this is not true. The dogma is that sequence determines structure; how, then, can very different sequences fold to the same structure? Here, we employ high temperature unfolding simulations to probe the pathways and specific interactions that direct the folding and unfolding of the SH3 domain. The SH3 metafold in the Dynameomics Database consists of 753 proteins with the same structure, but varied sequences and functions. To investigate the relationship between sequence and structure, we selected 17 targets from the SH3 metafold with high sequence variability. Six unfolding simulations were performed for each target, transition states were identified, revealing two general folding/unfolding pathways at the transition state. Transition states were also expressed as mathematical graphs of connected chemical nodes, and it was found that three positions within the structure, independent of sequence, were consistently more connected within the graph than any other nearby positions in the sequence. These positions represent a hub connecting different portions of the structure. Multiple sequence alignment and covariation analyses also revealed certain positions that were more conserved due to packing constraints and stabilizing long‐range contacts. This study demonstrates that members of the SH3 domain with different sequences can unfold through two main pathways, but certain characteristics are conserved regardless of the sequence or unfolding pathway. While sequence determines structure, we show that disparate sequences can provide similar interactions that influence folding and lead to similar structures. [ABSTRACT FROM AUTHOR]

Key points: Skeletal muscle relaxation has been primarily studied by assessing the kinetics of force decay. Little is known about the resultant dynamics of structural changes in myosin heads during relaxation.The naturally occurring nucleotide 2‐deoxy‐ATP (dATP) is a myosin activator that enhances cross‐bridge binding and kinetics.X‐ray diffraction data indicate that with elevated dATP, myosin heads were extended closer to actin in relaxed muscle and myosin heads return to an ordered, resting state after contraction more quickly.Molecular dynamics simulations of post‐powerstroke myosin suggest that dATP induces structural changes in myosin heads that increase the surface area of the actin‐binding regions promoting myosin interaction with actin, which could explain the observed delays in the onset of relaxation.This study of the dATP‐induced changes in myosin may be instructive for determining the structural changes desired for other potential myosin‐targeted molecular compounds to treat muscle diseases. Here we used time‐resolved small‐angle X‐ray diffraction coupled with force measurements to study the structural changes in FVB mouse skeletal muscle sarcomeres during relaxation after tetanus contraction. To estimate the rate of myosin deactivation, we followed the rate of the intensity recovery of the first‐order myosin layer line (MLL1) and restoration of the resting spacing of the third and sixth order of meridional reflection (SM3 and SM6) following tetanic contraction. A transgenic mouse model with elevated skeletal muscle 2‐deoxy‐ATP (dATP) was used to study how myosin activators may affect soleus muscle relaxation. X‐ray diffraction evidence indicates that with elevated dATP, myosin heads were extended closer to actin in resting muscle. Following contraction, there is a slight but significant delay in the decay of force relative to WT muscle while the return of myosin heads to an ordered resting state was initially slower, then became more rapid than in WT muscle. Molecular dynamics simulations of post‐powerstroke myosin suggest that dATP induces structural changes in myosin that increase the surface area of the actin‐binding regions, promoting myosin interaction with actin. With dATP, myosin heads may remain in an activated state near the thin filaments following relaxation, accounting for the delay in force decay and the initial delay in recovery of resting head configuration, and this could facilitate subsequent contractions. Key points: Skeletal muscle relaxation has been primarily studied by assessing the kinetics of force decay. Little is known about the resultant dynamics of structural changes in myosin heads during relaxation.The naturally occurring nucleotide 2‐deoxy‐ATP (dATP) is a myosin activator that enhances cross‐bridge binding and kinetics.X‐ray diffraction data indicate that with elevated dATP, myosin heads were extended closer to actin in relaxed muscle and myosin heads return to an ordered, resting state after contraction more quickly.Molecular dynamics simulations of post‐powerstroke myosin suggest that dATP induces structural changes in myosin heads that increase the surface area of the actin‐binding regions promoting myosin interaction with actin, which could explain the observed delays in the onset of relaxation.This study of the dATP‐induced changes in myosin may be instructive for determining the structural changes desired for other potential myosin‐targeted molecular compounds to treat muscle diseases. [ABSTRACT FROM AUTHOR]

The p53 protein is a commonly studied cancer target because of its role in tumor suppression. Unfortunately, it is susceptible to mutation‐associated loss of function; approximately 50% of cancers are associated with mutations to p53, the majority of which are located in the central DNA‐binding domain. Here, we report molecular dynamics simulations of wild‐type (WT) p53 and 20 different mutants, including a stabilized pseudo‐WT mutant. Our findings indicate that p53 mutants tend to exacerbate latent structural‐disruption tendencies, or vulnerabilities, already present in the WT protein, suggesting that it may be possible to develop cancer therapies by targeting a relatively small set of structural‐disruption motifs rather than a multitude of effects specific to each mutant. In addition, α‐sheet secondary structure formed in almost all of the proteins. α‐Sheet has been hypothesized and recently demonstrated to play a role in amyloidogenesis, and its presence in the reported p53 simulations coincides with the recent re‐consideration of cancer as an amyloid disease. [ABSTRACT FROM AUTHOR]

Abstract: Metamorphic proteins are biomolecules prone to adopting alternative conformations. Because of this feature, they represent ideal systems to investigate the general rules allowing primary structure to dictate protein topology. A comparative molecular dynamics study was performed on the denatured states of two proteins, sharing nearly identical amino‐acid sequences (88 %) but different topologies, namely an all‐α‐helical bundle protein named GA88 and an α+β‐protein named GB88. The analysis allowed successful design of and experimental validation of a site‐directed mutant that promotes, at least in part, the switch in folding from GB88 to GA88. The mutated position, in which a glutamic acid was replaced by a glutamine, does not make any intramolecular interactions in the native state of GA88, such that its stabilization can be explained by considering the effects on the denatured state. The results represent a direct demonstration of the role of the denatured state in sculpting native structure. [ABSTRACT FROM AUTHOR]

Prion diseases are associated with the misfolding of the prion protein (PrP) from its normal cellular form (Pr PC) to its infectious scrapie form (Pr PSc). Post-translational modifications in PrP in vivo can play an important role in modulating the process of misfolding. To gain more insight into the effects of post-translational modifications in PrP structure and dynamics and to test the hypothesis that such modifications can interact with the protein, we have performed molecular dynamics simulations of diglycosylated human Pr PC bound to a lipid bilayer via a glycophosphatidylinositol anchor. Multiple simulations were performed at three different pH ranges to explore pH effects on structure and dynamics. In contrast to simulations of protein-only Pr PC, no large effects were observed upon lowering the pH of the system. The protein tilted toward the membrane surface in all of the simulations and the putative Pr PSc oligomerization sites became inaccessible, thereby offering a possible protective mechanism against Pr PSc-induced misfolding of PrPC. [ABSTRACT FROM AUTHOR]

Myosin activation is a viable approach to treat systolic heart failure. We previously demonstrated that striated muscle myosin is a promiscuous ATPase that can use most nucleoside triphosphates as energy substrates for contraction. When 2-deoxy ATP (dATP) is used, it acts as a myosin activator, enhancing cross-bridge binding and cycling. In vivo, we have demonstrated that elevated dATP levels increase basal cardiac function and rescues function of infarcted rodent and pig hearts. Here we investigate the molecular mechanism underlying this physiological effect. We show with molecular dynamics simulations that the binding of dADP.Pi (dATP hydrolysis products) to myosin alters the structure and dynamics of the nucleotide binding pocket, myosin cleft conformation, and actin binding sites, which collectively yield a myosin conformation that we predict favors weak, electrostatic binding to actin. In vitro motility assays at high ionic strength were conducted to test this prediction and we found that dATP increased motility. These results highlight alterations to myosin that enhance cross-bridge formation and reveal a potential mechanism that may underlie dATP-induced improvements in cardiac function. [ABSTRACT FROM AUTHOR]

ABSTRACT Amyloids have long been associated with protein dysfunction and neurodegenerative diseases, but recent research has demonstrated that some organisms utilize the unique properties of the amyloid fold to create functional structures with important roles in biological processes. Additionally, new engineering approaches have taken advantage of amyloid structures for implementation in a wide variety of materials and devices. In this review, the role of amyloid in human disease is discussed and compared to the functional amyloids, which serve a largely structural purpose. We then consider the use of amyloid constructs in engineering applications, including their utility as building blocks for synthetic biology and molecular engineering. Biotechnol. Bioeng. 2017;114: 7-20. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Protein function is intimately linked to protein structure and dynamics yet experimentally determined structures frequently omit regions within a protein due to indeterminate data, which is often due protein dynamics. We propose that atomistic molecular dynamics simulations provide a diverse sampling of biologically relevant structures for these missing segments (and beyond) to improve structural modeling and structure prediction. Here we make use of the Dynameomics data warehouse, which contains simulations of representatives of essentially all known protein folds. We developed novel computational methods to efficiently identify, rank and retrieve small peptide structures, or fragments, from this database. We also created a novel data model to analyze and compare large repositories of structural data, such as contained within the Protein Data Bank and the Dynameomics data warehouse. Our evaluation compares these structural repositories for improving loop predictions and analyzes the utility of our methods and models. Using a standard set of loop structures, containing 510 loops, 30 for each loop length from 4 to 20 residues, we find that the inclusion of Dynameomics structures in fragment-based methods improves the quality of the loop predictions without being dependent on sequence homology. Depending on loop length, ∼25-75% of the best predictions came from the Dynameomics set, resulting in lower main chain root-mean-square deviations for all fragment lengths using the combined fragment library. We also provide specific cases where Dynameomics fragments provide better predictions for NMR loop structures than fragments from crystal structures. Online access to these fragment libraries is available at . [ABSTRACT FROM AUTHOR]

Integrative structural biology attempts to model the structures of protein complexes that are challenging or intractable by classical structural methods (due to size, dynamics, or heterogeneity) by combining computational structural modeling with data from experimental methods. One such experimental method is chemical crosslinking mass spectrometry (XL-MS), in which protein complexes are crosslinked and characterized using liquid chromatography-mass spectrometry to pinpoint specific amino acid residues in close structural proximity. The commonly used lysine-reactive N-hydroxysuccinimide ester reagents disuccinimidylsuberate (DSS) and bis(sulfosuccinimidyl)suberate (BS3) have a linker arm that is 11.4 Å long when fully extended, allowing Cα (alpha carbon of protein backbone) atoms of crosslinked lysine residues to be up to ∼24 Å apart. However, XL-MS studies on proteins of known structure frequently report crosslinks that exceed this distance. Typically, a tolerance of ∼3 Å is added to the theoretical maximum to account for this observation, with limited justification for the chosen value. We used the Dynameomics database, a repository of high-quality molecular dynamics simulations of 807 proteins representative of diverse protein folds, to investigate the relationship between lysine-lysine distances in experimental starting structures and in simulation ensembles. We conclude that for DSS/BS3, a distance constraint of 26-30 Å between Cα atoms is appropriate. This analysis provides a theoretical basis for the widespread practice of adding a tolerance to the crosslinker length when comparing XL-MS results to structures or in modeling. We also discuss the comparison of XL-MS results to MD simulations and known structures as a means to test and validate experimental XL-MS methods. [ABSTRACT FROM AUTHOR]

Membership in a protein domain database does not a domain make; a feature we realized when generating a consensus view of protein fold space with our consensus domain dictionary (CDD). This dictionary was used to select representative structures for characterization of the protein dynameome: the Dynameomics initiative. Through this endeavor we rejected a surprising 40% of the 1,695 folds in the CDD as being non-autonomous folding units. Although some of this was due to the challenges of grouping similar fold topologies, the dissonance between the cataloguing and structural qualification of protein domains remains surprising. Another potential factor is previously overlooked intrinsic disorder; predictions suggest that 40% of proteins have either local or global disorder. One thing is clear, filtering a structural database and ensuring a consistent definition for protein domains is crucial, and caution is prescribed when generalizations of globular domains are drawn from unfiltered protein domain datasets. [ABSTRACT FROM AUTHOR]

Using molecular dynamics simulations and steady-state fluorescence spectroscopy, we have identified a conformational change in the active site of a thermophilic flavoenzyme, NADH oxidase from Thermus thermophilus HB8 (NOX). The enzyme's far-UV circular dichroism spectrum, intrinsic tryptophan fluorescence, and apparent molecular weight measured by dynamic light scattering varied little between 25 and 75°C. However, the fluorescence of the tightly bound FAD cofactor increased approximately fourfold over this temperature range. This effect appears not to be due to aggregation, unfolding, cofactor dissociation, or changes in quaternary structure. We therefore attribute the change in flavin fluorescence to a temperature-dependent conformational change involving the NOX active site. Molecular dynamics simulations and the effects of mutating aromatic residues near the flavin suggest that the change in fluorescence results from a decrease in quenching by electron transfer from tyrosine 137 to the flavin. Proteins 2012. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

We have recently completed systematic molecular dynamics simulations of 807 different proteins representing 95% of the known autonomous protein folds in an effort we refer to as Dynameomics. Here we focus on the analysis of side chain conformations and dynamics to create a dynamic rotamer library. Overall this library is derived from 31,000 occurrences of each of 86,217 different residues, or 2.7 × 10 rotamers. This dynamic library has 74% overlap of rotamer distributions with rotamer libraries derived from static high-resolution crystal structures. Seventy-five percent of the residues had an assignable primary conformation, and 68% of the residues had at least one significant alternate conformation. The average correlation time for switching between rotamers ranged from 22 ps for Met to over 8 ns for Cys; this time decreased 20-fold on the surface of the protein and modestly for dihedral angles further from the main chain. Side chain S axis order parameters were calculated and they correlated well with those derived from NMR relaxation experiments ( R = 0.9). Relationships relating the S axis order parameters to rotamer occupancy were derived. Overall the Dynameomics rotamer library offers a comprehensive depiction of side chain rotamer preferences and dynamics in solution, and more realistic distributions for dynamic proteins in solution at ambient temperature than libraries derived from crystal structures, in particular charged surface residues are better represented. Details of the rotamer library are presented here and the library itself can be downloaded at . [ABSTRACT FROM AUTHOR]

The prion protein (PrP) is responsible for several fatal neurodegenerative diseases via conversion from its normal to disease-related isoform. The recombinant form of the protein is typically studied to investigate the conversion process. This constructs lacks the co- and post-translational modifications present in vivo, there the protein has two N-linked glycans and is bound to the outer leaflet of the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. The inherent flexibility and heterogeneity of the glycans, the plasticity of the GPI anchor, and the localization of the protein in a membrane make experimental structural characterization of biological constructs of cellular prion protein (PrPC) challenging. Yet this characterization is central in determining not only the suitability of recombinant (rec)-PrPC as a model for biological forms of the protein but also the potential role of co- and post-translational modifications on the disease process. Here, we present molecular dynamics simulations of three human prion protein constructs: (i) a protein-only construct modeling the recombinant form, (ii) a diglycosylated and soluble construct, and (iii) a diglycosylated and GPI-anchored construct bound to a lipid bilayer. We found that glycosylation and membrane anchoring do not significantly alter the structure or dynamics of PrPC, but they do appreciably modify the accessibility of the polypeptide surface PrPC. In addition, the simulations of membrane-bound PrPC revealed likely recognition domains for the disease-initiating PrPC:PrPSc (infectious and/or misfolded form of the prion protein) binding event and a potential mechanism for the observed inefficiency of conversion associated with differentially glycosylated PrP species. [ABSTRACT FROM AUTHOR]

Structure is only the first step in understanding the interactions and functions of proteins. In this paper, we explore the flexibility of proteins across a broad database of over 250 solvated protein molecular dynamics simulations in water for an aggregate simulation time of ∼6 μs. These simulations are from our Dynameomics project, and these proteins represent approximately 75% of all known protein structures. We employ principal component analysis of the atomic coordinates over time to determine the primary axis and magnitude of the flexibility of each atom in a simulation. This technique gives us both a database of flexibility for many protein fold families and a compact visual representation of a particular protein's native-state conformational space, neither of which are available using experimental methods alone. These tools allow us to better understand the nature of protein motion and to describe its relationship to other structural and dynamical characteristics. In addition to reporting general properties of protein flexibility and detailing many dynamic motifs, we characterize the relationship between protein native-state flexibility and early events in thermal unfolding and show that flexibility predicts how a protein will begin to unfold. We provide evidence that fold families have conserved flexibility patterns, and family members who deviate from the conserved patterns have very low sequence identity. Finally, we examine novel aspects of highly inflexible loops that are as important to structural integrity as conventional secondary structure. These loops, which are difficult if not impossible to locate without dynamic data, may constitute new structural motifs. [ABSTRACT FROM AUTHOR]

The folding of WW domains is rate limited by formation of a β-hairpin comprising residues from strands 1 and 2. Residues in the turn of this hairpin have reported Φ-values for folding close to 1 and have been proposed to nucleate folding. High Φ-values do not necessarily imply that the energetics of formation are a driving force for initiating folding. We demonstrate by NMR studies and molecular dynamics simulations that the first turn of the hYAP, FBP28, and PIN1 WW domains is structurally dynamic and solvent exposed in the native and folding transition states. It is, therefore, unlikely that the formation of the β-turn per se provides the energetic driving force for hairpin folding. It is more likely that the turn acts as an easily formed hinge that facilitates the formation of the hairpin; it is a nucleus as defined by the nucleation-condensation mechanism whereby a diffuse nucleus is stabilized by associated interactions. [ABSTRACT FROM AUTHOR]

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

We have determined consensus protein-fold classifications on the basis of three classification methods, SCOP, CATH, and Dali. These classifications make use of different methods of defining and categorizing protein folds that lead to different views of protein-fold space. Pairwise comparisons of domains on the basis of their fold classifications show that much of the disagreement between the classification systems is due to differing domain definitions rather than assigning the same domain to different folds. However, there are significant differences in the fold assignments between the three systems. These remaining differences can be explained primarily in terms of the breadth of the fold classifications. Many structures may be defined as having one fold in one system, whereas far fewer are defined as having the analogous fold in another system. By comparing these folds for a nonredundant set of proteins, the consensus method breaks up broad fold classifications and combines restrictive fold classifications into metafolds, creating, in effect, an averaged view of fold space. This averaged view requires that the structural similarities between proteins having the same metafold be recognized by multiple classification systems. Thus, the consensus map is useful for researchers looking for fold similarities that are relatively independent of the method used to compare proteins. The 30 most populated metafolds, representing the folds of about half of a nonredundant subset of the PDB, are presented here. The full list of metafolds is presented on the Web. [ABSTRACT FROM AUTHOR]

The conformational equilibrium between 310- and α-helical structure has been studied via high-resolution NMR spectroscopy by Millhauser and coworkers using the MW peptide Ac-AMAAKAWAAKA AAARA-NH2. Their 750-MHz nuclear Overhauser effect spectroscopy (NOESY) spectra were interpreted to reflect appreciable populations of 310-helix throughout the peptide, with the greatest contribution at the N and C termini. The presence of simultaneous αN (i,i + 2) and αN (i,i + 4) NOE cross-peaks was proposed to represent conformational averaging between 310- and α-helical structures. In this study, we describe 25-nsec molecular dynamics simulations of the MW peptide at 298 K, using both an 8 Å and a 10 Å force-shifted nonbonded cutoff. The ensemble averages of both simulations are in reasonable agreement with the experimental helical content from circular dichroism (CD), the 3JHNα coupling constants, and the 57 observed NOEs. Analysis of the structures from both simulations revealed very little formation of contiguous i → i + 3 hydrogen bonds (310-helix); however, there was a large population of bifurcated i → i + 3 and i → i + 4 α-helical hydrogen bonds. In addition, both simulations contained considerable populations of π-helix ( i → i + 5 hydrogen bonds). Individual turns formed over residues 1-9, which we predict contribute to the intensities of the experimentally observed αN (i,i + 2) NOEs. Here we show how sampling of both folded and unfolded structures can provide a structural framework for deconvolution of the conformational contributions to experimental ensemble averages. [ABSTRACT FROM AUTHOR]

We examined the hydration of amides of α3D, a simple, designed three-helix bundle protein. Molecular dynamics calculations show that the amide carbonyls on the surface of the protein tilt away from the helical axis to interact with solvent water, resulting in a lengthening of the hydrogen bonds on this face of the helix. Water molecules are bonded to these carbonyl groups with partial occupancy (∼50%-70%), and their interaction geometries show a large variation in their hydrogen bond lengths and angles on the nsec time scale. This heterogeneity is reflected in the carbonyl stretching vibration (amide I′ band) of a group of surface Ala residues. The surface-exposed amides are broad, and shift to lower frequency (reflecting strengthening of the hydrogen bonds) as the temperature is decreased. By contrast, the amide I′ bands of the buried 13C-labeled Leu residues are significantly sharper and their frequencies are consistent with the formation of strong hydrogen bonds, independent of temperature. The rates of hydrogen-deuterium exchange and the proton NMR chemical shifts of the helical amide groups also depend on environment. The partial occupancy of the hydration sites on the surface of helices suggests that the interaction is relatively weak, on the order of thermal energy at room temperature. One unexpected feature that emerged from the dynamics calculations was that a Thr side chain subtly disrupted the helical geometry 4-7 residues N-terminal in sequence, which was reflected in the proton chemical shifts and the rates of amide proton exchange for several amides that engage in a mixed 310/α/π-helical conformation. [ABSTRACT FROM AUTHOR]

The effects of engineered disulfide bonds on protein stability are poorly understood because they can influence the structure, dynamics, and energetics of both the native and denatured states. To explore the effects of two engineered disulfide bonds on the stability of barnase, we have conducted a combined molecular dynamics and NMR study of the denatured state of the two mutants. As expected, the disulfide bonds constrain the denatured state. However, specific extended β-sheet structure can also be detected in one of the mutant proteins. This mutant is also more stable than would be predicted. Our study suggests a possible cause of the very high stability conferred by this disulfide bond: the wild-type denatured ensemble is stabilized by a nonnative hydrophobic cluster, which is constrained from occurring in the mutant due to the formation of secondary structure. [ABSTRACT FROM AUTHOR]

Nine nonnative conformations of ubiquitin, generated during two different thermal denaturation trajectories, were simulated under nearly native conditions (62 °C). The simulations included all protein and solvent atoms explicitly, and simulation times ranged from 1-2.4 ns. The starting structures had α-carbon root-mean-square deviations (RMSDs) from the crystal structure of 4-12 Å 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 Å 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 Å 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 Å 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. [ABSTRACT FROM AUTHOR]

Photographs of several conformational protein molecules are presented.