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A widely held hypothesis regarding the thermostability of thermophilic proteins states asserts that, at any given temperature, thermophilic proteins are more rigid than their mesophilic counterparts. Many experimental and computational studies have addressed this question with conflicting results. Here, we compare two homologous enzymes, one mesophilic (Escherichia coli FMN-dependent nitroreductase; NTR) and one thermophilic (Thermus thermophilus NADH oxidase; NOX), by multiple molecular dynamics simulations at temperatures from 5 to 100 degrees C. We find that the global rigidity/flexibility of the two proteins, assessed by a variety of metrics, is similar on the time scale of our simulations. However, the thermophilic enzyme retains its native conformation to a much greater degree at high temperature than does the mesophilic enzyme, both globally and within the active site. The simulations identify the helix F-helix G 'arm' as the region with the greatest difference in loss of native contacts between the two proteins with increasing temperature. In particular, a network of electrostatic interactions holds helix F to the body of the protein in the thermophilic protein, and this network is absent in the mesophilic counterpart.

All-atom molecular dynamics (MD) simulations on increasingly powerful computers have been combined with experiments to characterize protein folding in detail over wider time ranges. The folding of small ultrafast folding proteins is being simulated on micros timescales, leading to improved structural predictions and folding rates. To what extent is 'closing the gap' between simulation and experiment for such systems providing insights into general mechanisms of protein folding?

Both folded and unfolded conformations should be observed for a protein at its melting temperature (T(m)), where DeltaG between these states is zero. In an all-atom molecular dynamics simulation of chymotrypsin inhibitor 2 (CI2) at its experimental T(m), the protein rapidly loses its low-temperature native structure; it then unfolds before refolding to a stable, native-like conformation. The initial unfolding follows the unfolding pathway described previously for higher-temperature simulations: the hydrophobic core is disrupted, the beta-sheet pulls apart and the alpha-helix unravels. The unfolded state reached under these conditions maintains a kernel of structure in the form of a non-native hydrophobic cluster. Refolding simply reverses this path, the side-chain interactions shift, the helix refolds, and the native packing and hydrogen bonds are recovered. The end result of this refolding is not the initial crystal structure; it contains the proper topology and the majority of the native contacts, but the structure is expanded and the contacts are long. We believe this to be the native state at elevated temperature, and the change in volume and contact lengths is consistent with experimental studies of other native proteins at elevated temperature and the chemical denaturant equivalent of T(m).

The human gene for catechol O-methyltransferase (COMT) contains a common polymorphism that results in substitution of methionine (M) for valine (V) at residue 108 of the soluble form of the protein. While the two proteins have similar kinetic properties, 108M COMT loses activity more rapidly than 108V COMT at 37 degrees C. The cosubstrate S-adenosylmethionine (SAM) stabilizes the activity of 108M COMT at 40 degrees C. The 108M allele has been associated with increased risk for breast cancer, obsessive-compulsive disorder, and aggressive and highly antisocial manifestations of schizophrenia. In the current work, we have constructed homology models for both human COMT polymorphs and performed molecular dynamics simulations of these models at 25, 37, and 50 degrees C to explore the structural consequences of the 108V/M polymorphism. The simulations indicated that replacing valine with the larger methionine residue led to greater solvent exposure of residue 108 and heightened packing interactions between M108 and helices alpha2, alpha4 (especially with R78), and alpha5. These altered packing interactions propagated subtle changes between the polymorphic site and the active site 16 A away, leading to a loosening of the active site. At physiological temperature, 108M COMT sampled a larger distribution of conformations than 108V. 108M COMT was more prone to active-site distortion and had greater overall, and SAM binding site, solvent accessibility than 108V COMT at 37 degrees C. Similar structural perturbations were observed in the 108V protein only at 50 degrees C. Addition of SAM tightened up the cosubstrate pocket in both proteins and prevented the altered packing at the polymorphic site in 108M COMT.

The self-assembly of beta(2)-microglobulin into fibrils leads to dialysis-related amyloidosis. pH-mediated partial unfolding is required for the formation of the amyloidogenic intermediate that then self-assembles into amyloid fibrils. Two partially folded intermediates of beta(2)-microglobulin have been identified experimentally and linked to the formation of fibrils of distinct morphology, yet it remains difficult to characterize these partially unfolded states at high resolution using experimental approaches. Consequently, we have performed molecular dynamics simulations at neutral and low pH to determine the structures of these partially unfolded amyloidogenic intermediates. In the low-pH simulations, we observed the formation of alpha-sheet structure, which was first proposed by Pauling and Corey. Multiple simulations were performed, and two distinct intermediate state ensembles were identified that may account for the different fibril morphologies. The predominant early unfolding intermediate was nativelike in structure, in agreement with previous NMR studies. The late unfolding intermediate was significantly disordered, but it maintained an extended elongated structure, with hydrophobic clusters and residual alpha-extended chain strands in specific regions of the sequence that map to amyloidogenic peptides. We propose that the formation of alpha-sheet facilitates self-assembly into partially unfolded prefibrillar amyloidogenic intermediates.

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 (PrP(C)) to its infectious scrapie isoform (PrP(Sc)), which causes neurodegenerative diseases in many mammals. These two forms have identical sequences and are conformational isomers, with heightened formation of beta-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 beta-sheet and by addition of a portion of the N-terminus to widen the sheet by another 2-3 strands.

Abstract Protein folding/unfolding is a complicated process that defies high-resolution characterization by experimental methods. As an alternative, atomistic molecular dynamics simulations are now routinely employed to elucidate and magnify the accompanying conformational changes and the role of solvent in the folding process. However, the level of detail necessary to map the process at high spatial–temporal resolution provides an overwhelming amount of data. As more and better tools are developed for analysis of these large data sets and validation of the simulations, one is still left with the problem of visualizing the results in ways that provide insight into the folding/unfolding process. While viewing and interrogating static crystal structures has become commonplace, more and different approaches are required for dynamic, interconverting, unfolding, and refolding proteins. Here we review a variety of approaches, ranging from straightforward to complex and unintuitive for multiscale analysis and visualization of protein folding and unfolding. Graphical abstract Unlabelled Image Highlights • Examination of protein folding and unfolding pathways via several different methods scaling from the atomistic level to whole-structure and multi-structure levels • Visualizing folding in multiple ways provides a more comprehensive picture of specific interactions that lead to different conformational states. [ABSTRACT FROM AUTHOR]

d-amino acids are useful building blocks for de novo peptide design and they play a role in aging-related diseases associated with gradual protein racemization. For amino acids with achiral side chains, one should be able to presume that the conformational propensities of l- and d-amino acids are a reflection of one another due to the straightforward geometric inversion at the Cα atom. However, this presumption does not account for the directionality of the backbone dipole and the inverted propensities have never been definitively confirmed in this context. Furthermore, there is little known of how alternative side chain chirality affects the backbone conformations of isoleucine and threonine. Using a GGXGG host–guest pentapeptide system, we have completed exhaustive sampling of the conformational propensities of the d-amino acids, including d-allo-isoleucine and d-allo-threonine, using atomistic molecular dynamics simulations. Comparison of these simulations with the same systems hosting the cognate l-amino acids verifies that the intrinsic backbone conformational propensities of the d-amino acids are the inverse of their cognate l-enantiomers. Where amino acids have a chiral center in their side chain (Thr, Ile) the β-configuration affects the backbone sampling, which in turn can confer different biological properties. [ABSTRACT FROM PUBLISHER]

The classification of protein folds is necessarily based on the structural elements that distinguish domains. Classification of protein domains consists of two problems: the partition of structures into domains and the classification of domains into sets of similar structures (or folds). Although similar topologies may arise by convergent evolution, the similarity of their respective folding pathways is unknown. The discovery and the characterization of the majority of protein folds will be followed by a similar enumeration of available protein folding pathways. Consequently, understanding the intricacies of structural domains is necessary to understanding their collective folding pathways. We review the current state of the art in the field of protein domain classification and discuss methods for the systematic and comprehensive study of protein folding across protein fold space via atomistic molecular dynamics simulation. Finally, we discuss our large-scale Dynameomics project, which includes simulations of representatives of all autonomous protein folds. [ABSTRACT FROM AUTHOR]

All atom molecular dynamics simulations have become a standard method for mapping equilibrium protein dynamics and non-equilibrium events like folding and unfolding. Here, we present detailed methods for performing such simulations. Generic protocols for minimization, solvation, simulation, and analysis derived from previous studies are also presented. As a measure of validation, our water model is compared with experiment. An example of current applications of these methods, simulations of the ultrafast folding protein Engrailed Homeodomain are presented including the experimental evidence used to verify their results. Ultrafast folders are an invaluable tool for studying protein behavior as folding and unfolding events measured by experiment occur on timescales accessible with the high-resolution molecular dynamics methods we describe. Finally, to demonstrate the prospect of these methods for folding proteins, a temperature quench simulation of a thermal unfolding intermediate of the Engrailed Homeodomain is described. [Copyright &y& Elsevier]

We have assessed the published predictions of the pathway of folding of the B domain of protein A, the pathway most studied by computer simulation. We analyzed the transition state for folding of the three-helix bundle protein, by using experimental φ values on some 70 suitable mutants. Surprisingly, the third helix, which has the most stable α-helical structure as a peptide fragment, is poorly formed in the transition state, especially at its C terminus. The protein folds around a nearly fully formed central helix, which is stabilized by extensive hydrophobic side chain interactions. The turn connecting the poorly structured first helix to the central helix is unstructured, but the turn connecting the central helix to the third is in the process of being formed as the N-terminal region of the third helix begins to coalesce. The transition state is inconsistent with a classical framework mechanism and is closer to nucleation-condensation. None of the published atomistic simulations are fully consistent with the experimental picture although many capture important features. There is a continuing need for combining simulation with experiment to describe folding pathways, and of continued testing to improve predictive methods. [ABSTRACT FROM AUTHOR]

Combining experimental and simulation data to describe all of the structures and the pathways involved in folding a protein is problematical. Transition states can be mapped experimentally by φ values, but the denatured state is very difficult to analyse under conditions that favour folding. Also computer simulation at atomic resolution is currently limited to about a microsecond or less. Ultrafast-folding proteins fold and unfold on timescales accessible by both approaches, so here we study the folding pathway of the three-helix bundle protein Engrailed homeodomain. Experimentally, the protein collapses in a microsecond to give an intermediate with much native α-helical secondary structure, which is the major component of the denatured state under conditions that favour folding. A mutant protein shows this state to be compact and contain dynamic, native-like helices with unstructured side chains. In the transition state between this and the native state, the structure of the helices is nearly fully formed and their docking is in progress, approximating to a classical diffusion–collision model. Molecular dynamics simulations give rate constants and structural details highly consistent with experiment, thereby completing the description of folding at atomic resolution. [ABSTRACT FROM AUTHOR]

Explores mapping of the early steps in the pH-induced conformational conversion of the prion protein (PrP). Factors that trigger conversion of the PrP; Effect of acidic pH on PrP conformation; Characterization of change at the atomic level.

Photographs of several conformational protein molecules are presented.

The principle of microscopic reversibility states that at equilibrium the number of molecules entering a state by a given path must equal those exiting the state via the same path under identical conditions or, in structural terms, that the conformations along the two pathways are the same. There has been some indirect evidence indicating that protein folding is such a process, but there have been few conclusive findings. In this study, we performed molecular dynamics simulations of an ultrafast unfolding and folding protein at its melting temperature to observe, on an atom-by-atom basis, the pathways the protein followed as it unfolded and folded within a continuous trajectory. In a total of 0.67 µs of simulation in water, we found six transient denaturing events near the melting temperature (323 and 330 K) and an additional refolding event following a previously identified unfolding event at a high temperature (373 K). In each case, unfolding and refolding transition state ensembles were identified, and they agreed well with experiment on the basis of a comparison of S and Φ values. On the basis of several structural properties, these 13 transition state ensembles agreed very well with each other and with four previously identified transition states from high-temperature denaturing simulations. Thus, not only were the unfolding and refolding transition states part of the same ensemble, but in five of the seven cases, the pathway the protein took as it unfolded was nearly identical to the subsequent refolding pathway. These events provide compelling evidence that protein folding is a microscopically reversible process. In the other two cases, the folding and unfolding transition states were remarkably similar to each other but the paths deviated. [ABSTRACT FROM AUTHOR]

Recent work suggests that some native conformations of proteins can vary with temperature. To obtain an atomic-level description of this structural and conformational variation, we have performed all-atom; explicit-solvent molecular dynamics simulations of bovine pancreatic ribonuclease A (RNase A) up to its melting temperature (Tm ≈ 337 K). RNase A has a thermal pretransition near 320 K [Stelea, S. D., Pancoska, P., Benight, A. S., and Keiderling, T. A. (2001) Protein Sci. 10, 970-978]. Our simulations identify a conformational change that coincides with this pretransition. Between 310 and 320 K, there is a small but significant decrease in the number of native contacts, β-sheet hydrogen bonding, and deviation of backbone conformation from the starting structure, and an increase in the number of nonnative contacts. Native contacts are lost in β-sheet regions and in α1, partially due to movement of α1 away from the β-sheet core. At 330 and 340 K, a nonnative helical segment of residues 15-20 forms, corresponding to a helix observed in the N-terminal domain-swapped dimer [Liu, Y. S., Hart, P. J., Schulnegger, M. P., and Eisenberg, D. (1998) Proc. Nail. Acad. Sci. U.S.A. 95, 3437-3432]. The conformations observed at the higher temperatures possess nativelike topology and overall conformation, with many native contacts, but they have a disrupted active site. We propose that these conformations may represent the native state at elevated temperature, or the N' state. These simulations show that subtle, functionally important changes in protein conformation can occur below the Tm. [ABSTRACT FROM AUTHOR]

Abstract: Two independent replica-exchange molecular dynamics (REMD) simulations with an explicit water model were performed of the Trp-cage mini-protein. In the first REMD simulation, the replicas started from the native conformation, while in the second they started from a nonnative conformation. Initially, the first simulation yielded results qualitatively similar to those of two previously published REMD simulations: the protein appeared to be over-stabilized, with the predicted melting temperature 50–150K higher than the experimental value of 315K. However, as the first REMD simulation progressed, the protein unfolded at all temperatures. In our second REMD simulation, which starts from a nonnative conformation, there was no evidence of significant folding. Transitions from the unfolded to the folded state did not occur on the timescale of these simulations, despite the expected improvement in sampling of REMD over conventional molecular dynamics (MD) simulations. The combined 1.42μs of simulation time was insufficient for REMD simulations with different starting structures to converge. Conventional MD simulations at a range of temperatures were also performed. In contrast to REMD, the conventional MD simulations provide an estimate of Tm in good agreement with experiment. Furthermore, the conventional MD is a fraction of the cost of REMD and continuous, realistic pathways of the unfolding process at atomic resolution are obtained. [Copyright &y& Elsevier]

Molecular dynamics simulations can be used to reveal the detailed conformational behaviors of peptides and proteins. By comparing fragment and full-length protein simulations, we can investigate the role of each peptide segment in the folding process. Here, we take advantage of information regarding the helix formation process from our previous simulations of barnase and protein A as well as new simulations of four helical fragments from these proteins at three different temperatures, starting with both helical and extended structures. Segments with high helical propensity began the folding process by tethering the chain through side chain interactions involving either polar interactions, such as salt bridges, or hydrophobic staples. These tethers were frequently nonnative (i.e., not i→i + 4 spacing) and provided a scaffold for other residues, thereby limiting the conformational search. The helical structure then propagated on both sides of the tether. Segments with low stability and propensity formed later in the folding process and utilized contacts with other portions of the protein when folding. These helices formed via a tertiary contact-assisted mechanism, primarily via hydrophobic contacts between residues distant in sequence. Thus, segments with different helical propensities appear to play different roles during protein folding. Furthermore, the active role of nonlocal side chains in helix formation highlights why we must move beyond simple hierarchical models of protein folding. [ABSTRACT FROM AUTHOR]

There is a change from three-state to two-state kinetics of folding across the homeodomain superfamily of proteins as the mechanism slides from framework to nucleation-condensation. The tendency for framework folding in this family correlates with inherent helical propensity. The cellular myeloblastis protein (c-Myb) falls in the mechanistic transition region. An earlier, preliminary report of protein engineering experiments and molecular dynamics simulations (MD) showed that the folding mechanism for this protein has aspects of both the nucleation-condensation and framework models. In the more in-depth analysis of the MD trajectories presented here, we find that folding may be attributed to both of these mechanisms in different regions of the protein. The folding of the loop, middle helix, and turn is best described by nucleation-condensation, whereas folding of the N and C-terminal helices may be described by the framework model. Experimentally, c-Myb folds by apparent two-state kinetics, but the MD simulations predict that the kinetics hide a high-energy intermediate. We stabilized this hypothetical folding intermediate by deleting a residue (P174) in the loop between its second and third helices, and the mutant intermediate is long-lived in the simulations. Equilibrium and kinetic experiments demonstrate that folding of the ΔP174 mutant is indeed three-state. The presence and shape of the intermediate observed in the simulations were confirmed by small angle X-ray scattering experiments. [Copyright &y& Elsevier]

We have analysed the transition state of folding of the four-helix FF domain from HYPA/FBP11 by high-resolution experiment and simulation as part of a continuing effort to understand the principles of folding and the refinement of predictive methods. The major transition state for folding was subjected to a Φ-value analysis utilising 50 mutants. The transition state contained a nucleus for folding centred around the end of helix 1 (H1) and the beginning of helix 2 (H2). Secondary structure in this region was fully formed (ΦF=0.9–1) and tertiary interactions were well developed. Interactions in the distal part of the native structure were weak (ΦF=0–0.2). The hydrophobic core and other parts of the protein displayed intermediate Φ-values, suggesting that interactions coalesce as the end of H1 and beginning of H2 are in the process of being formed. The distribution of Φ-values resembled that of barnase, which folds via an intermediate, rather than that of CI2 which folds by a concerted nucleation–condensation mechanism. The overall picture of the transition state structure identified in molecular dynamics simulations is in qualitative agreement, with the turn connecting H1 and H2 being formed, a loosened core, and H4 partially unfolded and detached from the core. There are some differences in the details and interpretation of specific Φ-values. [Copyright &y& Elsevier]