MOLECULAR-DYNAMICS SIMULATIONS OF PROTEIN UNFOLDING AND LIMITED REFOLDING - CHARACTERIZATION OF PARTIALLY UNFOLDED STATES OF UBIQUITIN IN 60-PERCENT METHANOL AND IN WATER

Extensive experimental data are available on the native, partially and fully unfolded states of ubiquitin. Two and three-dimensional NMR exp eriments of a partially unfolded form of the protein in 60% methanol i ndicate that approximately one-half of the molecule contains disrupted but native-like structure while the other half is unstructured and/or contains non-native structure. In contrast, the interpretation of hyd rogen-exchange data have led to the conclusion that this state is nati ve-like. Thus, there are discrepancies between the experimental studie s, or interpretations based on the data. We compare the results of mol ecular dynamics simulations, under varying conditions, with the experi mental results. The simulations extend past 0.5 ns and include explici t solvent molecules: either pure water or 60% methanol. To begin with, ubiquitin was thermally denatured in water (at 498 K). Two particular structures, or ''aliquots'', during the unfolding process were select ed for further study (60 and 198 ps). These structures were then simul ated separately in water and 60% methanol at a lower and experimentall y meaningful temperature (335 K). The conformations generated from the structure extracted later in the simulation contained significant amo unts of non-native structure in the presence of methanol while satisfy ing both the NMR and hydrogen exchange data. Tn fact, clearly non-nati ve regions of the structure yielded the desired protection from hydrog en exchange. In contrast, an earlier, more native-like, intermediate d id not do as well at predicting the hydrogen-exchange behavior and was inconsistent with the NMR data. These data suggest that the results a nd interpretations using the different experimental techniques can be reconciled by a single state. This finding also brings into question t he practice of interpreting protection to hydrogen exchange in terms o f native secondary and tertiary structure, especially when one has wea k patterns and low protection factors. When the partially unfolded sta tes were placed in pure water, the protein collapsed and began to refo ld. Therefore, the desired solvent-dependent properties were observed: the partially unfolded conformations with increased exposure of hydro phobic residues remained expanded in methanol but collapsed in water a s the non-polar groups minimized their exposure to solvent.