Vadim V. Mozhaev

High pressure effects on protein structure and function

Many biochemists would regard pressure as a physical parameter mainly of theoretical interest and of rather limited value in experimental biochemistry. The goal of this overview is to show that pressure is a powerful tool for the study of proteins and modulation of enzymatic activity.

Exploiting the effects of high hydrostatic pressure in biotechnological applications

Abstract Applying hydrostatic pressure to biological systems and processes can alter their characteristics. In addition to its use as a basic research tool for investigating the kinetics and thermodynamics of biological systems at the molecular level, the application of pressure is also being used to modify the properties of biological materials to preserve or improve their qualities. This article reviews the principles underlying the observed effects of applied pressure on biological systems, and discusses current and potential application of pressure in biotechnological processes.

Application of high hydrostatic pressure for increasing activity and stability of enzymes

Elevated hydrostatic pressure has been used to increase catalytic activity and thermal stability of alpha-chymotrypsin (CT). For an anilide substrate, characterized by a negative value of the reaction activation volume (DeltaV( not equal)), an increase in pressure at 20 degrees C results in an exponential acceleration of the hydrolysis rate catalyzed by CT reaching a 6.5-fold increase in activity at 4700 atm (4.7 kbar). Due to a strong temperature dependence of DeltaV( not equal), the acceleration effect of high pressure becomes more pronounced at high temperatures. For example, at 50 degrees C, under a pressure of 3.6 kbar, CT shows activity which is more than 30 times higher than the activity at normal conditions (20 degrees C, 1 atm). At pressures of higher than 3.6 kbar, the enzymatic activity is decreased due to a pressure-induced denaturation.Elevated hydrostatic pressure is also efficient for increasing stability of CT against thermal denaturation. For example, at 55 degrees C, CT is almost instantaneously inactivated at atmospheric pressure, whereas under a pressure of 1.8 kbar CT retains its anilide-hydrolyzing activity during several dozen minutes. Additional stabilization can be achieved in the presence of glycerol, which is most effective for protection of CT at an intermediate concentration of 40% (v/v). There has been observed an additivity in stabilization effects of high pressure and glycerol: thermal inactivation of pressure-stabilized CT can be decelerated in a supplementary manner by addition of 40% (v/v) glycerol. The protection effect of glycerol on the catalytic activity and stability of CT becomes especially pronounced when both extreme factors of temperature and pressure reach critical values. For example, at approximately 55 degrees C and 4.7 kbar, enzymatic activity of CT in the presence of 40% (v/v) glycerol is severalfold higher than in aqueous buffer.The results of this study are discussed in terms of the hypotheses which explain the action of external and medium effects on protein structure, such as preferential hydration and osmotic pressure.

Structure-stability relationships in proteins: new approaches to stabilizing enzymes

Abstract A comparison of the structure of (a) proteins from thermophilic and mesophilic microorganisms, (b) closely related proteins with different thermostability from various mesophilic sources, and (c) mutationally altered enzymes with those from wild strains has been carried out. The main molecular mechanisms existing in nature for the creation of thermostable proteins have been elucidated. The most important mechanism is the strengthening of hydrophobic interactions in the interior of the protein globule. This mechanism has been employed to advance a novel approach to enzyme stabilization which consists of the following steps. A protein is first made to unfold into a random coil-like state and then the folding of the protein is performed in one of three ways: (1) in ‘non-native’ conditions, (2) in the presence of substances which can interact with the protein in a noncovalent fashion, (3) after covalent modification of the unfolded protein.

The principles of enzyme stabilization. II. Increase in the thermostability of enzymes as a result of multipoint noncovalent interaction with a polymeric support.

Abstract The catalytic activity, thermostability (resistance to monomolecular thermo-inactivation) and molecular mobility of chymotrypsin and trypsin mechanically entrapped into polymethacrylate and polyacrylamide gels have been studied. It has been established that the thermostability of the enzymes does not depend on the concentration of electroneutral polyacrylamide gel over the range of 0–50 w/w%. However, in polymethacrylate gel of concentration higher than 30 w/w%, when a high catalytic activity is retained, the thermostability of chymotrypsin dramatically increases: in 50 w/w% gel the first-order rate constant for thermoinactivation of the enzyme at 60°C is 10−5 that in water. Based on these data and also on experimentally obtained results on transitional and rotational diffusion of both native and modified enzymes, the following mechanism of enzyme stabilization is formulated and proved. In principle, the protein molecule of an enzyme may form with the three-dimensional lattice of polyelectrolyte gel multiple noncovalent linkages (via electrostatic or hydrogen bonds); as a result, the structure of the enzyme becomes more rigid and its thermostability should increase. However, since these bonds are relatively weak, in diluted gels they can hardly be realized, as the “quenching” of the transitional movement of the enzyme molecules, accompanying complex formation would have required a heavy entropy loss. At the same time, in concentrated gels, this unfavourable entropy contribution is absent as the polymers lattice provides significant steric hindrances for the transitional diffusion, so that the molecules almost stop moving. That is why weak linkages between the protein globule and the support can be realized here. That the complex formation does take place is indicated by the fact the rotational diffusion of the protein molecules is almost completely frozen. When there is no specific protein-support interaction (in polyacrylamide gel), no deceleration of the rotational movement of the protein molecules occurs and no noticeable increase in the thermostability of the enzymes is observed. It is possible that the mechanism discovered by us functions in vivo and is responsible for the stability (and, which is important, for stability regulation) of the proteins incorporated in biomembranes. On the other hand, the results obtained by us may enrich enzyme engineering, as they allow the general strategy of production of stabilized enzymes to be outlined.

Strategy for Stabilizing Enzymes Part One: Increasing Stability of Enzymes via their Multi-Point Interaction with a Support

This review states that the covalent multi-point attachment of enzymes to a support is the most general approach to stabilize them against different denaturing conditions, namely against their inactivation caused by protein unfolding. It is suggested that the change in the wavelength of the maximum emission in fluorescence spectra of a protein, resulting from its denaturation, can be used to evaluate a priori the effectiveness of stabilization. The copolymerization method of enzyme immobilization, as the most promising approach to stabilizing enzymes, is discussed in detail.

Structure-stability relationship in proteins; fundamental tasks and strategy for the development of stabilized enzyme catalysts for biotechnology

The problem of relationships between the protein structure and its stability comprises two major questions. First, how to elucidate the peculiarities of the protein structure responsible for its stability. Second, knowing the general molecular basis of protein stability, how to change the structure of a given protein in order to increase its stability. This review is an attempt to show the modern state of the first (fundamental) and the second (applied) aspects of the problem.

Enzyme-polyelectrolyte complexes in water-ethanol mixtures: Negatively charged groups artificially introduced into α-chymotrypsin provide additional activation and stabilization effects

Formation of noncovalent complexes between alpha-chymotrypsin (CT) and a polyelectrolyte, polybrene (PB), has been shown to produce two major effects on enzymatic reactions in binary mixtures of polar organic cosolvents with water. (i) At moderate concentrations of organic cosolvents (10% to 30% v/v), enzymatic activity of CT is higher than in aqueous solutions, and this activation effect is more significant for CT in complex with PB (5- to 7-fold) than for free enzyme (1.5- to 2.5-fold). (ii) The range of cosolvent concentrations that the enzyme tolerates without complete loss of catalytic activity is much broader. For enhancement of enzyme stability in the complex with the polycation, the number of negatively charged groups in the protein has been artificially increased by using chemical modification with pyromellitic and succinic anhydrides. Additional activation effect at moderate concentrations of ethanol and enhanced resistance of the enzyme toward inactivation at high concentrations of the organic solvent have been observed for the modified preparations of CT in the complex with PB as compared with an analogous complex of the native enzyme. Structural changes behind alterations in enzyme activity in water-ethanol mixtures have been studied by the method of circular dichroism (CD). Protein conformation of all CT preparations has not changed significantly up to 30% v/v of ethanol where activation effects in enzymatic catalysis were most pronounced. At higher concentrations of ethanol, structural changes in the protein have been observed for different forms of CT that were well correlated with a decrease in enzymatic activity. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 267-277, 1997.

Relationship between surface hydrophilicity of a protein and its stability against denaturation by organic solvents

The stability of α‐chymotrypsin covalently modified with a strongly hydrophilic modifier, pyromellitic dianhydride, against solvent‐induced denaturation in water—organic solvent binary mixtures has been studied. It was found that the hydrophilization results in a strong stabilization of the enzyme against denaturation by organic solvents. The stabilizing effect is explained in terms of the enhanced ability of the hydrophilized enzyme to keep its hydration shell, which is indispensable for supporting the native protein conformation, from denaturing stripping by organic solvents

Regioselective enzymatic acylation as a tool for producing solution-phase combinatorial libraries

Abstract A simple combinatorial strategy for sequential regioselective enzymatic acylation of multifunctional lead compounds has been developed and demonstrated using a polyhydroxylated flavonoid, bergenin, as a model. The approach is based on the ability of different enzymes to regioselectively acylate different sites on a lead molecule without affecting other similar functional groups. In sharp contrast to enzymatic acylation, conventional chemical acylation methods showed almost complete lack of regioselectivity. The enzymatic strategy was applied successfully to produce a solution phase combinatorial library of 167 distinct selectively acylated derivatives of bergenin on a robotic workstation in a 96-well plate format. General applicability of the automated combinatorial biocatalysis strategy is discussed.