Protein Crystal Growth Under High Pressure

Abstract

In this chapter, I would like to describe following two main roles of high pressure (up to 250 MPa) on protein crystal growth. 1. High pressure as a tool for enhancing crystallization of a protein 2. High pressure as a tool for modifying a three-dimensional (3D) structure of a protein molecule For the first role, Visuri et al. reported that the total amount of obtained crystals of glucose isomerase (GI) was drastically increased with increasing pressure, for the first time (Visuri et al., 1990). Such drastic enhancements probably play an important role in increasing the success rate of 3D structure analysis of protein molecules, since crystallization is still the rate limiting step in the structure analysis process. Although they were the pioneers of this field, they did not do further studies on the growth mechanisms of GI crystals. After their pioneer work, many studies have been done on solubility (Section 2), nucleation (Section 3), and growth kinetics (Section 4) under high pressure. Here I would like to review and classify these studies, and present the potential of high pressure as a tool for enhancing protein crystallization. For the second role, Kundrot & Richards were the pioneers of this field. They analyzed 3D structure of hen egg-white lysozyme under high pressure at the atomic level, for the first time (Kundrot & Richards, 1987). High-pressure protein crystallography is a prerequisite to understanding effects of pressure on an enzymatic activity of a protein at the atomic level (Makimoto et al., 1984). The structural information also plays an important role in the studies of deep-sea organisms (Yayanos, 1986). Pressure probably influences the protein structure through the structure of surrounding water molecules. Thus the protein structure under high pressure has to be solved with water of hydration at ambient temperatures, since a flash cooling method obviously influences the crystal structure (Charron et al., 2002); a freezing process of the method probably influences the structure of the surrounding water molecules, and the process prevents us from an “in situ” analysis of the protein structure with water of hydration. In addition, mainly due to the technical difficulties, total number of studies on high-pressure protein crystallography is not so many at this stage. In Section 5, I would like to review the studies on “in situ” protein structure analysis under high pressure, and present a new methodology for an ideal “in situ” structure analysis.