MOLECULAR DYNAMICS SIMULATIONS OF PROTEIN UNFOLDING

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The immediate goal of the proposed research is to perform realistic molecular modeling studies relating to protein folding. Although much is known of the structural details of the native folded conformation of proteins, very little is known about the actual folding process. This process has important implications for many biological processes: protein degradation, protein translocation, aging and human diseases, including cancer and amyloid diseases. Determination of the structural characteristics of intermediate states during folding are crucial for understanding the mechanism of these processes. However, the cooperative nature of folding generally results in only minute amounts of partially folded intermediates at equilibrium. Furthermore, even when relatively stable intermediates are isolated, they are difficult to characterize structurally because of the increased motion and lack of fixed structure throughout the molecules. Given that protein folding is of such widespread importance to human health and that experimental approaches provide only limited amounts of information on the structural transitions and interactions occurring during protein folding, computer simulation methods provide an avenue for elucidating the molecular details of the process. Our approach is to perform molecular dynamics simulations where the protein is "denatured" in solution. With this method, the events occurring as the protein unfolds and any intermediates in the process can be characterized at the atomic level. It is unlikely that experimental approaches will ever be able to provide structural information about these intermediates and the folding process in comparable detail to that available for native proteins. Therefore, this is an area where experiment needs theory. But, the simulations must always be compared to experiment to ensure that they are relevant. Consequently, alpha-lytic protease, and chymotrypsin inhibitor 2, and cytochrome b5 were chosen for study, as experimental studies of the folding behavior of these proteins are being pursued actively and collaborations with these experimentalists have begun. The results of preliminary simulations of all three proteins are in good agreement with experiment and suggest that these simulation studies are worth pursuing. The proposed simulations should provide detailed dynamic, molecular models of folding intermediates that go beyond, but are complementary to, what can be garnered using experimental methods. Furthermore, the nature of the transition state of unfolding is being explored with CI2. In addition, a comparison of the unfolding properties of these two proteins should allow determination of the general forces acting during protein folding that are independent of the model system chosen. These simulations also address what distinguishes a molten globule (e.g. as adopted by alpha-lytic protease) from other folding intermediates (e.g., apocytochrome b5). After investigating these and other systems, we will be in a better position to apply these techniques to proteins directly involved in diseases for which there are less experimental data available.