Darwin O.V. Alonso and Valerie Daggett. (1995) J. Mol. Biol. 247, 501-520.
Extensive experimental data are available on the native, partially and fully unfolded states of ubiquitin. Two and three- dimensional NMR experiments of a partially unfolded form of the protein in 60% methanol indicate that approximately one-half of the molecule contains disrupted but native-like structure while the other half is unstructured and/or contain non-native structure. In contrast the interpretation of hydrogen-exchange data has let to the conclusion that this state is native-like. Thus, there are discrepancies between the experimental studies, or between interpretations based on the data. We compare the results of molecular dynamics simulation, under varying conditions, with the experimental results. The simulation extend past 0.5 ns and include explicit solvent molecules: either pure water or 60% methanol. To begin with, ubiquitin was thermally denatured in water (498K). Two particular structures from the unfolding trajectory (from 60 and 198 ps) were then selected for further study. These structures were then simulated separately in water and in 60% methanol at a lower and experimentally meaningful temperature (335 K 62 C). The conformations generated from the structure extracted later in the unfolding simulation contained significant amounts of non-native structure in the presence of methanol while satisfying both the NMR figure and hydrogen exchange data. Non-native regions of the simulated later structure yielded the experimental patterns of protection from hydrogen exchange figure. In contrast, the earlier, more native-like intermediate did not do as well at predicting the experimental protection from hydrogen exchange and was inconsistent with the NMR date. These data suggest that the results and interpretations using the different experimental techniques can be reconciled by a single state. This finding also brings into question the practice of interpreting protection to hydrogen exchange in terms of native secondary and tertiary structure, especially where one has weak patterns and low protection factors. When the partially unfolded states were placed in pure water, the protein collapsed and began to refold. Therefore, the desired solvent-dependent properties were observed: the partially unfolded conformations with increased exposure of hydrophobic residues remained expanded in methanol but collapsed in water as the non-polar groups minimized their exposure to solvent.
Refs:
Briggs and Roder. (1992) PNAS. 89, 2017. Kinetics of ubiquitin folding, NMR.
Pan and Briggs. (1992) Biochem. 31, 11405. Hydrogen exchange in 60% MeOH.
Cox et al. (1993) J. Mol. Biol. 234, 483. NMR of ubiquitin fragments.
Harding et al. (1991) Biochem. 31, 3120. 2D-NMR of ubiquitin in 60% MeOH.
