Extensively hydrated but folded: a novel state of globular proteins stabilized at high pressure and low temperature.

We studied conformational fluctuations of the transcription factor c-Myb R2 subdomain (52 residues with three Trp) at high pressure and low temperature (5°C) using two different spectroscopic methods, Trp fluorescence and (1)H NMR, on its chemically stable mutant C130I (pseudo-wild-type (WT(S))), which has a large internal cavity. As pressure was increased from 3 to 300 MPa, the Trp fluorescence λ(max) of WT(S) shifted from 342 to ∼355 nm, clearly showing that the three Trp rings become fully exposed to the polar environment, which usually is taken to indicate that the protein underwent unfolding. In contrast, as pressure was increased from 3 to 300 MPa, the high-field-shifted (1)H NMR signals characteristic of the folded state showed a still higher-field shift, but no significant changes in their intensity. The last result unequivocally shows that the protein remains largely folded at 300 MPa. The apparent discrepancy between the two predictions would only be solved if one were to postulate the existence of an extensively hydrated but folded state in WT(S). Intriguingly, such a state was not found in a cavity-filling mutant of WT(S), C130I/V103L, suggesting that this state is mediated by cavity hydration. The generality and significance of this state in proteins are discussed.