Pressure-Tuning Spectroscopy of Proteins: Fourier Transform Infrared Studies in the Diamond Anvil Cell

Abstract

The effect of hydrostatic pressure on the secondary structure of proteins can be followed by Fourier transform infrared (FTIR) spectroscopy in the diamond anvil cell. Pressure-induced changes in the amide I’ region of the deconvolved spectrum are used to follow the features of the secondary structure up to 20 kbar. The changes in the side chains such as tyrosine also can be followed. A self-deconvolution and fitting procedure is presented that allows the determination of both pressure-induced and temperature-induced changes in the secondary structure of proteins. The method takes into account the elastic, as well as the possible conformational, effects on the spectral bands of the protein. Applications are presented on pressure-induced changes in several proteins. Attention is also given to the influence of inert cosolvents. The fundamental principles of the phase diagram of proteins are presented to clarify their importance for understanding the behavior of proteins under pressure at different temperatures. Our results show that the infrared technique explores unique aspects of the behavior of proteins under these extreme conditions. The study of the effects of pressure has received considerable attention in recent years (Balny et al., 1992; Silva & Weber, 1993). In general, low pressures induce reversible changes such as the dissociation of protein-protein complexes, the binding of ligands, and conformational changes. Pressures higher than about 5 kbar induce denaturation, which in most cases is irreversible. However, reports on a few proteins indicate that such high pressures may also cause reversible changes. One such protein is horse scrum albumin (Chen & Heremans, 1990). A molecular interpretation of these phenomenon is based on the fact that pressure mainly affects the volume of a system, thus damping the molecular fluctuations. Temperature effects are known to affect both the kinetic energy and the volume of the system. Early in this century, it was shown that one can cook an egg by subjecting it to high pressure (Bridgman, 1914). The appearance of the pressure-induced coagulum of egg white is quite different from the coagulum induced by temperature.