Putting on the Squeeze: Solution NMR Investigations of Protein Structure and Hydration under High Pressure

Abstract

It is well known that high hydrostatic pressures can induce the unfolding of proteins. The physical underpinnings of this phenomenon have been investigated extensively but remain controversial. Changes in solvation energetics due to applied hydrostatic pressure have been a commonly proposed mechanism for unfolding, but recent studies have provided strong evidence that elimination of void volumes in the native folded state is a principal determinant. Here we use the cavity-containing L99A mutant of T4 lysozyme to examine the pressure unfolding of a multi-domain protein using solution NMR. The cavity-containing C-terminal domain completely unfolds at moderate pressures while the N-terminal domain remains largely structured to high pressures. This pressure response is completely suppressed by benzene binding to the hydrophobic cavity. These results contrast to the pseudo wild type protein, which has a residual cavity volume very similar to that of the L99A-benzene complex but shows extensive subglobal reorganizations with pressure. Encapsulation of the L99A mutant in the aqueous nanoscale core of a reverse micelle suppresses the pressure-induced unfolding transition due to the volume restriction and promotes high-pressure filling of the cavity with water. This result indicates that hydration of the hydrophobic cavity is more energetically unfavorable than global unfolding. Overall these observations point to a range of cooperativity and energetics in the pressure response of proteins and illuminate the fact that small changes in physical parameters can significantly alter this response. Supported by NSF grant MCB- 115803 and by NIH postdoctoral fellowship GM087099 to N.V.N.