Our goal is to perform realistic molecular dynamics (MD) simulations, in concert with experiment, to characterize the pathway of protein folding/unfolding at atomic resolution. Although much is known of the structural details of the native, folded state of proteins, very little is known about partially folded conformations and the details of the folding/unfolding process. Unfolding has important implications for many biological processes, including amyloid diseases. Detailed knowledge of the folding process should also lead to improved structure prediction algorithms. In pursuit of this goal, the original and renewed proposals were to perform high-temperature MD simulations of the unfolding of small globular proteins. This endeavor has been successful with respect to establishing the methods, devising ways to identify and test models of transition states, developing analysis tools for the evaluation of residual structure in partially unfolded and denatured proteins, and developing approaches for comparison of MD results with experiment. But, there are still issues that need to be addressed. There has been a huge difference between the experimental time scale of protein unfolding and what is computationally feasible. As a result, past studies employed harsh conditions to destabilize the native state, typically very high temperature. Now it is possible to run longer simulations at experimentally relevant temperatures, particularly for newly discovered ultrafast folding (-1-15 gs) and unfolding (N5-10 ns) proteins. Consequently, this proposal focuses on these ultrafast folding/unfolding proteins, in particular various 3-helix bundles (the engrailed homeodomain, En-HD; protein A; and a3D, a de novo designed protein) and small 2- and 3-stranded [B-structures (trpzip and WW domains) (Specific Aim 1). In addition, simulations of chemically induced denaturation (urea and guanidinium chloride) and the effect of introducing stabilizing osmolytes (trimethylamine N-oxide) will be investigated. (Specific Aim 2), as the molecular basis of solvent-induced conformational behavior remains unknown. To investigate the detailed determinants of folding for simple helical and [B- structures, simulations will be performed of natural sequence variants within the En-HD and WW domain superfamilies. (Specific Aim 3). With a new, parallelized simulation program under development, much larger systems can be investigated, and 'test tube' simulations will be pursued with multiple copies of the protein to investigate the effect of neighboring molecules (crowding) on the folding/unfolding process. Because this new program also makes it possible to perform microsecond simulations in reasonable periods of time, refolding of the MD-denatured ultrafast proteins, by lowering the temperature or diluting out the denaturant, will be attempted. (Specific Aim 4). Concurrent experimental studies on these systems are being pursued by our collaborators: Professors Alan Fersht and William DeGrado.
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