Structural and Thermodynamic Characterization of T4 Lysozyme ...

The characterization of non-native states stabilized under a variety of conditions is important towardsunderstanding how proteins fold into their biologically functional, native structures (2). It is widelybelieved that the dominant driving force in protein folding is the hydrophobic effect and thatdenaturation can be described as the transfer of hydrophobic residues to water. Although thehydrophobic compound transfer model (3-6) largely succeeds in explaining the thermodynamic stabilityof proteins as a function of temperature, it does not explain denaturation with pressure (7). In particular,this model fails to explain the magnitude and pressure dependence of the volume difference between thenative and denatured states. Recent simulation studies and experimental work suggest that this failure isdue to fundamental differences between the temperature- and pressure-denatured states of a protein (1,8-12). These studies suggest that unlike thermally and chemically denatured states, the pressuredenaturedstate is one in which water penetration into the protein is favorable, and that a significantcontribution to the volume reduction with pressure is the hydration of internal cavities or packingdefects.The literature raises specific questions that require further study. Is pressure denaturation consistentwith the water penetration model? How does the volume change upon denaturation correlate with cavityvolume? Can other hydration mechanisms be distinguished from cavity filling? What constitutes apressure-denatured state? We attempt to answer these questions by characterizing the pressuredenaturedstates of several mutants of the protein T4 lysozyme with varying cavity sizes.T4 lysozyme is a small globular protein, 164 amino acid residues in length, with a molecular weightof 18.6 kDa (Figure 1 (f)) (13). Over 300 X-ray structures of T4 lysozyme mutants have been depositedin the Protein Data Bank, and the thermal stabilities of many of these mutants have been measured (14).Pressure-induced water filling of an enlarged hydrophobic cavity in the L99A mutant of the pseudowild-typeT4 lysozyme (Figure 1 (b), cavity 6) has been observed by X-ray crystallography (1).Molecular dynamics simulations suggested that four water molecules cooperatively fill this cavity as theapplied hydrostatic pressure is increased (1). No water molecules were observed in the correspondingcavity of the pseudo-wild-type, WT* (Figure 1 (a), cavity 6) (1).3