High-pressure as a tool to study some proteins’ properties: conformational modification, activity and oligomeric dissociation

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Abstract

Hydrostatic pressure, as temperature, constitutes an efficient physical parameter to modify equilibrium and rate of biological processes. In this review, we will not present all the implications of high-pressure, but we will focus on proteins’ structural conformational modification, unfolding, oligomeric or protein aggregates dissociation and enzymatic activity. To this aim, some optical methods that were used in association with high-pressure to study protein structures (i.e. fourth derivative absorbance spectroscopy, fluorescence spectroscopy, infrared spectroscopy) and protein activities (kinetics measurements: stopped-flow and pressure jump methods) will be described in details. Some applications of these methods will be given including effects on proteins of food interest.

Introduction

The effects of an applied hydrostatic pressure (or high-pressure) on biological systems appear to constitute an interesting tool for their study. As a thermodynamic parameter, hydrostatic pressure was known for many years to act on biological materials in a similar but not identical way as temperature. However, it was disregarded for a long time by biochemists mainly because all the basic concepts (and the thermodynamics) were focused on the chemistry involved and because general ideas on what pressure can add to the understanding of the proteins’ behavior were lacking. In the recent decades, technological progress in the field of physics have shown, along with parameters such as temperature and solvent conditions, that pressure can be used for a more detailed thermodynamic and kinetics description of biological processes and regulation of their behavior. The effects of pressure on a number of proteins, nucleoproteins and membranes were recently reviewed (Groß & Jaenicke, 1994, Mozhaev, Heremans, Frank, Masson & Balny, 1996, Balny, Masson & Heremans, 2002), including biotechnological applications (Balny, Hayashi, Heremans & Masson, 1992, Mozhaev, Heremans, Frank, Masson & Balny, 1994, Hayashi & Balny, 1996, Hayashi, 2002, Heremans, 1997, Knorr, 1999, Konczewicz, Balny & Cheftel, 2000). This review will present the most recent aspects of experimental development using high hydrostatic pressure applied to proteins and will be illustrated with some new data.

Section snippets

Pressure effects on biological systems

The general behavior of biological systems under high hydrostatic pressure is governed by Le Chatelier's principle, which predicts that the application of pressure shifts an equilibrium towards a state that occupies a smaller volume, and accelerates processes for which the transition state has a smaller volume than the ground state. Therefore, pressure favors processes that are accompanied by negative volume changes. Unfortunately, in contrast to simple chemical reactions, it is very difficult

Pressure vs. temperature effects

Closely associated with the Le Chatelier's principle is the principle of microscopic ordering, which states that, at constant temperature, an increase in pressure increases the degree of ordering of molecules resulting in a decrease in entropy of the system.

Studies of the temperature–pressure relationship effects on proteins were performed many years ago by Hawley (1971) who showed an antagonistic effect of these two physical parameters through a phase diagram of protein denaturation (Fig. 1).

Pressure effects on protein functions

Pressure modifies the rate of enzyme–catalyzed reactions via changes in the structure of an enzyme or changes in the reaction mechanism. The first theoretical basis explaining the responses of enzymatic activities to high hydrostatic pressures was formulated, by Laidler, in 1950 (Laidler, 1950) but a more recent overview concerning this phenomenon was given by Morild (1981) twenty years ago. The effects of pressure on the overall reaction may be the consequence of its effects on the reaction

Recent progress

Table 1 shows that major biophysical methods used routinely at atmospheric pressure, except circular dichroism, have been successfully adapted to high-pressure conditions (Mozhaev et al., 1996; Taniguchi, Stanley & Ludwig, 2001). Some of these methods have been recently used and developed in our laboratory to study enzyme kinetics or protein conformation and will be described in the present paper.

The 33-kDa protein from spinach photosystem II particle

In the laboratory, Ruan et al. (2001a) have recently studied the 33-kDa protein isolated from the spinach photosystem II particle that constitutes an ideal model to explore high-pressure protein unfolding. This protein has a very low free energy, as previously reported by chemical unfolding studies, suggesting that its unfolding transition must be easy to modulate by rather mild pressure. It contains one tryptophan (Trp241) and 8 tyrosine residues that are convenient probes to follow protein

The problem of pressure dissociation and pressure aggregation

The mechanisms of pressure dissociation are complex as illustrated below with some recent examples already published.

The sweet potato β-amylase, an enzyme found in higher plants, is composed of four identical subunits—each containing six cysteine residues. Due to steric hindrance, these residues are not reactive to specific sulfhydryl reagent. Under high-pressure, enhancement of one of the cysteine reactivity with a specific sulfhydryl reagent was shown to be due to a local conformation

Biotechnological and biomedical applications

Progress in the application of high-pressure in biotechnology requires the development of both processes and equipment, together with refinement of the theory of pressure effects on biomolecules and biological systems. Fortunately, many technical problems have been solved and industrial large-scale equipment for high-pressure treatment are now commercially available (50 l capacity under pressure up to 500 MPa, semi-continuous system of 1–4 tons per h capacity for the treatment of liquid foods).

Conclusions and future prospects

If high-pressure has long been known to denature native proteins and dissociate oligomeric proteins, recent results showed that all the potentialities have not yet been explored, and that new area could be opened such as those concerning problems related to protein aggregation. We would like to point out that high-pressure, which induces reversible denaturation, is a real alternative way that can be associated with the classical use of chaotrope molecules to both induce and study protein's

Acknowledgements

We would like to thank warmly our colleagues who did, in our laboratory, the experimental work related in this review (the complete list is too long to be given) and for the very stimulati ng discussions that we had with them. This paper was written in part during an available years work of V. Lullien-Pellerin at the INSERM U128. The experimental results were partly obtained by financial supports from INRA, COST D10 network and grants from INSERM/Chinese Academy of Sciences.

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