Hydrophobic core formation and dehydration in protein folding studied by generalized-ensemble simulations

Despite its small size, chicken villin headpiece subdomain HP36 folds into the native structure with a stable hydrophobic core within several microseconds. How such a small protein keeps up its conformational stability and fast folding in solution is an important issue for understanding molecular mechanisms of protein folding. In this study, we performed multicanonical replica-exchange simulations of HP36 in explicit water, starting from a fully extended conformation. We observed at least five events of HP36 folding into nativelike conformations. The smallest backbone root mean-square deviation from the crystal structure was 1.1 A. In the nativelike conformations, the stably formed hydrophobic core was fully dehydrated. Statistical analyses of the simulation trajectories show the following sequential events in folding of HP36: 1), Helix 3 is formed at the earliest stage; 2), the backbone and the side chains near the loop between Helices 2 and 3 take nativelike conformations; and 3), the side-chain packing at the hydrophobic core and the dehydration of the core side chains take place simultaneously at the later stage of folding. This sequence suggests that the initial folding nucleus is not necessarily the same as the hydrophobic core, consistent with a recent experimental phi-value analysis.