Avant-propos et remerciements

Abstract

Constant pressure and temperature molecular dynamics techniques have been employed to investigate the changes in structure and volumes of two globular proteins, superoxide dismutase and lysozyme, under pressure. Compression (the relative changes in the proteins’ volumes), computed with the Voronoi technique, is closely related with the so-called protein intrinsic compressibility, estimated by sound velocity measurements. In particular, compression computed with Voronoi volumes predicts, in agreement with experimental estimates, a negative bound water contribution to the apparent protein compression. While the use of van der Waals and molecular volumes underestimates the intrinsic compressibilities of proteins, Voronoi volumes produce results closer to experimental estimates. Remarkably, for two globular proteins of very different secondary structures, we compute identical (within statistical error) protein intrinsic compressions, as predicted by recent experimental studies. Changes in the protein interatomic distances under compression are also investigated. It is found that, on average, short distances compress less than longer ones. This nonuniform contraction underlines the peculiar nature of the structural changes due to pressure in contrast with temperature effects, which instead produce spatially uniform changes in proteins. The structural effects observed in the simulations at high pressure can explain protein compressibility measurements carried out by f luorimetric and hole burning techniques. Finally, the calculation of the proteins static structure factor shows significant shifts in the peaks at short wavenumber as pressure changes. These effects might provide an alternative way to obtain information concerning compressibilities of selected protein regions. During the past few years, considerable effort has developed to understand the origin of the pressure effects on the structure and volume of proteins (1–3) and to elucidate the mechanism for protein denaturation at high pressure (4, 5). Many experimental techniques have been used to study pressure induced changes in proteins, but only in rare instances has computer simulation been employed. Here, we present a molecular dynamics (MD) investigation of the microscopic compression of two solvated proteins. By simulating these systems in the NPT ensemble at room temperature and at increasing pressures (from 0.1 to 2000 MPa), we were able to compute averages and fluctuations of the protein volumes and relate these microscopic properties to the experimental thermodynamic compressibilities. Experimentally, the effects of pressure on the protein structure are investigated by probing the corresponding changes in volume and by measuring compressibility, the latter being related to volume fluctuations, protein flexibility and, indirectly, protein functionality (6). Although crystallography (7), f luorescence spectroscopy (8), NMR (9), and hole burning (10) experiments have all been used to make estimates of protein compressibility, most of the available compressibility data come from sound velocity measurements (11, 12). Critical investigations of these experimental results have indicated that the compressibility of a protein can be divided into at least two components of opposite sign. Due to compressible cavities and voids in the protein interior, the first component of the protein intrinsic compressibility, bp, is positive. The remaining term, bHyd, is the contribution to the compressibility due to hydration and bound water. As the compressibility of single amino acids and small peptides in solution is negative (13), bHyd is thought to be negative. Most recent work (12) has indicated that the isothermal intrinsic compressibility of the protein, defined as the compressibility of the protein interior, is unique for all proteins and is about 3 times smaller than that of water. To distinguish, at a microscopic level, the protein intrinsic compressibility from contributions due to the solvents, the behavior of alternative definitions of protein compressions derived from computer simulation are juxtaposed here with the general experimental results. Although a few simulations of proteins at high pressure (14, 15) have been reported in the past, we carry out here a systematic correlation between experimental protein compressibilities and the simulation results. Additional issues concerning the intrinsic compressibility of proteins are raised by high pressure experiments on proteins carried out with techniques other than velocimetry. Although measurements of bp from hole burning experiments are in agreement with sound velocity estimates, f luorimetric determination of distances between trytophan and heme groups in different heme proteins have led to intrinsic compressibilities higher than that of water. In contrast, x-ray crystallography applied to atmospheric and high pressure crystals of lysozyme resulted in an overall bp that is one order of magnitude lower than that of water and one-third of that estimated from sound velocity experiments. Thus, an additional goal of this study is to address these experimental issues by characterizing the pressure effects on the protein interatomic distances derived from constant pressure MD simulations in light of the above experimental results. MATERIALS AND METHODS MD Simulations. All results reported in this paper were derived from a series ofMD simulations of solvated superoxide dismutase (SOD) and of the tetragonal crystal of hen egg white lysozyme at constant temperature and pressure, carried out at 300 K and 0.1, 1000, and 2000 MPa. Average pressures and temperature were kept constant using an extended Lagrangian method, the application of which to solvated protein has been discussed in ref. 16. The extended Lagrangian equations of The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: MD, molecular dynamics; SOD, superoxide dismutase; SPC, simple point charge model. *To whom reprint requests should be addressed.