Abstract
Nine nonnative conformations of ubiquitin, generated during two different thermal denaturation trajectories, were simulated under nearly native conditions (62 degrees C). The simulations included all protein and solvent atoms explicitly, and simulation times ranged from 1-2.4 ns. The starting structures had alpha-carbon root-mean-square deviations (RMSDs) from the crystal structure of 4-12 A and radii of gyration as high as 1.3 times that of the native state. In all but one case, the protein collapsed when the temperature was lowered and sampled conformations as compact as those reached in a control simulation beginning from the crystal structure. In contrast, the protein did not collapse when simulated in a 60% methanol:water mixture. The behavior of the protein depended on the starting structure: during simulation of the most native-like starting structures (<5 A RMSD to the crystal structure) the RMSD decreased, the number of native hydrogen bonds increased, and the secondary and tertiary structure increased. Intermediate starting structures (5-10 A RMSD) collapsed to the radius of gyration of the control simulation, hydrophobic residues were preferentially buried, and the protein acquired some native contacts. However, the protein did not refold. The least native starting structures (10-12 A RMSD) did not collapse as completely as the more native-like structures; instead, they experienced large fluctuations in radius of gyration and went through cycles of expansion and collapse, with improved burial of hydrophobic residues in successive collapsed states.