This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Background: The central paradigm of protein folding is the existence of a thermodynamically stable, native state in which amphiphilic polypeptides form unique three-dimensional structures with a closed-packed hydrophobic core. Based on an information theory model of hydrophobic interactions, we proposed that proteins stabilized by hydrophobic driving forces at ambient pressure are destabilized at elevated pressures due to water penetration into the hydrophobic core. To examine this proposition, we measured the reversible folding/unfolding transition of the protein staphylococcal nuclease at ambient temperature and kilobar pressures using small angle neutron scattering, and complemented these measurements with molecular dynamics (MD) simulations. Previous work: Previous simulations of pressure-induced protein unfolding have shown that the application of several kilobars of pressure in MD simulations slows protein conformational dynamics to such an extent that the observation times for unfolding become prohibitively large even for current high-performance computing capabilities. To circumvent this constraint, we combined random insertions of water molecules into the protein interior with MD simulations at progressively higher pressures to produce the onset of unfolding. In addition, we simulated multiple unfolding trajectories to obtain ensemble averages of protein conformational properties along the trajectories. The picture that emerges from our simulations is that high pressure in addition to water insertions into the hydrophobic core is required to induce protein unfolding, in agreement with our original information theory prediction. Proposed work: Since water insertions were artificially imposed in this study, a question that naturally arises is: What is the mechanism of water penetration into the protein interior at high pressure that leads to unfolding? To answer this question, we propose implementing the Serial Replica Exchange Molecular Dynamics (SREMD) method to enhance sampling of protein conformational space at high pressures. The SREMD method is comparable in efficiency to the replica exchange method, which has been shown to enhance sampling of protein conformational states, thereby circumventing sampling issues associated with the multiple time scales involved in protein folding. SREMD also runs asynchronously on distributed computing environments, and can be modified to incorporate more efficient treatments of explicit water solvent. The protein we have chosen for this study is the 36-residue villin headpiece subdomain (HP-36). Unlike the smaller (20 amino acid) alpha-helical peptide studied earlier, HP-36 folds into a well-defined, three-dimensional structure with a close-packed hydrophobic core. Moreover, it consists of only naturally occurring amino acids and requires no disulfide bonds to fold. The folding of HP-36 in explicit water from an unfolded state at high temperature has been observed previously in a one-microsecond MD simulation. The proposed SREMD simulations of HP-36 in explicit water over a range of temperatures at high pressure will allow sampling of protein conformations with significantly greater water penetration than has been possible up to now. We anticipate that the analysis of these simulations using recent advances in molecular theory of hydration will lead to new insights into the underlying mechanisms of pressure-induced protein unfolding, and will further test our prediction of the destabilization of the hydrophobic core due to water penetration at high pressures. To date, we have implemented the SREMD method in NAMD for HP-36 in explicit water (~25,000 atoms) at ambient and high pressures using a Linux cluster of 128 Intel Xeon EM64T 3.4 GHz dual processors maintained by the Ohio Supercomputing Center (OSC). The computing resources requested here will be used for a long production run on the system previously tested on this Linux cluster. We plan to apply for a larger (MRAC or LRAC) resource allocation in the next quarterly cycle based on the results of this simulation.
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