Katsuhiro Tamura

Protein crystallization under high pressure

Pressure is expected to be an important parameter to control protein crystallization, since hydrostatic pressure affects the whole system uniformly and can be changed very rapidly. So far, a lot of studies on protein crystallization have been done. Solubility of protein depends on pressure. For instance, the solubility of tetragonal lysozyme crystal increased with increasing pressure, while that of orthorhombic crystal decreased. The solubility of subtilisin increased with increasing pressure. Crystal growth rates of protein also depend on pressure. The growth rate of glucose isomerase was significantly enhanced with increasing pressure. The growth rate of tetragonal lysozyme crystal and subtilisin decreased with increasing pressure. To study the effects of pressure on the crystallization more precisely and systematically, hen egg white lysozyme is the most suitable protein at this stage, since a lot of data can be used. We focused on growth kinetics under high pressure, since extensive studies on growth kinetics have already been done at atmospheric pressure, and almost all of them have explained the growth mechanisms well. The growth rates of tetragonal lysozyme decreased with pressure under the same supersaturation. This means that the surface growth kinetics significantly depends on pressure. By analyzing the dependence of supersaturation on growth rate, it was found that the increase in average ledge surface energy of the two-dimensional nuclei with pressure explained the decrease in growth rate. At this stage, it is not clear whether the increase in surface energy with increasing pressure is the main reason or not. Fundamental studies on protein crystallization under high pressure will be useful for high pressure crystallography and high pressure protein science.

Stress tolerance of pressure-shocked Saccharomyces cerevisiae

Pressure shock treatment induced synthesis of heat shock protein (hsp104) and tolerance against various stresses such as high temperature, high pressure and high concentration of ethanol in Saccharomyces cerevisiae. The optimum pressures that induced maximal tolerance against these stresses were in the range of 50–75 MPa and depended on the type of stress. However, pressure shock did not stimulate trehalose production in the cells.

Effect of High-Pressure Gas on Yeast Growth

Microcalorimetry is a useful tool for monitoring the growth behavior of microorganisms. In this study, microcalorimetry was used to investigate the effects of nitrogen, air, oxygen, nitrous oxide, argon, and krypton at high pressure on the growth of the yeast Saccharomyces cerevisiae. Growth thermograms (metabolic heat vs. incubation time) were generated to estimate metabolic activity under compressed gases and to determine the 50% inhibitory pressure (IP50) and minimum inhibitory pressure (MIP), which are regarded as indices of the toxicity of compressed gases. Based on MIP values, the most toxic to the least toxic gases were found to be: O2 > N2O > air > Kr > N2 > Ar.

Effects of pressure on the phase transition of bilayers in liposomes influence of cholesterol and α-tocopherol

The effects of pressure on the gel-to-liquid crystalline phase transition temperature of dimyristoylphosphatidylcholine (DMPC) bilayers containing cholesterol, alpha-tocopherol, and alpha-tocopheryl acetate were studied by fluorescence depolarization. The transition temperature of cholesterol mixtures (greater than 7.5 mol%) was lower than that of 100% DMPC at atmospheric pressure, but it became higher than the latter on increase in pressure. The thermodynamic parameters of the transition (delta V, delta S, delta H) were estimated and the functions of cholesterol and alpha-tocopherols in the bilayers are discussed.

Measurement of Microbial Activities Under High Pressure by Calorimetry

Microcalorimetry is becoming an important method for study of the metabolic activities of cells and biological tissues. This method is mainly based on the fact that the heat evolved during metabolic processes is strictly proportional to the metabolic activity, so the magnitude of the calorimetrie signal is an index of biological activity. This method was adopted to investigate the thermotolerance of heat- and pressure-shocked yeast and to estimate microbial activities at high pressure. The effect of the non-reducing disaccharide trehalose on the stress response of yeast was also studied by calorimetry.

A statistical mechanical analysis of the effect of long-chain alcohols and high pressure upon the phase transition temperature of lipid bilayer membranes

Long-chain n-alcohols decrease the main phase-transition temperature of lipid vesicle membranes at low concentrations but increase it at high concentrations. The nonlinear phenomenon is unrelated to the interdigitation and is analyzed by assuming that alcohols form solid solutions with solid as well as liquid phases. The biphasic response originates from the balance of the free energy difference of alcohols in the liquid and solid membranes (delta gA) and the alcohol-lipid interaction free energy difference (delta u) between the two phases. When delta gA less than 0 and delta u greater than 0, or delta gA less than delta u less than 0, the transition temperature decreases monotonously according to the increase in the alcohol concentration. When delta gA greater than 0 and delta u less than 0, or delta gA greater than delta u greater than 0, it increases monotonously. Biphasic response occurs with a minimum temperature when delta u greater than delta gA greater than 0, and with a maximum temperature when delta u less than delta gA less than 0. When the alcohol carbon-chain length becomes closer to the lipid carbon-chain length, delta u is equalized by delta gA, and the temperature minimum of the main transition is shifted to extremely low alcohol concentrations. Hence, long-chain alcohols predominantly elevate the main transition temperature and lose their anesthetic potency. High pressure decreased both delta gA and delta u. Presumably, high pressure improves the packing efficiency of liquid membranes and decreases the difference between the solid and liquid membrane properties.

Solubility measurements of protein crystals under high pressure by in situ observation of steps on crystal surfaces

We successfully measured equilibrium temperatures Te of tetragonal hen egg-white lysozyme crystals under high pressure by in situ observation of steps on the {110} faces of the crystals. The dependence of Te on the concentration of lysozyme corresponds to that of the solubility Ce on temperature. The precision of the solubility determined in this study is significantly higher than that in previous works. One Te could be measured during short time less than 70 minutes. This method for solubility measurements of protein crystals under high pressure is the fastest one at this stage.

Hydrophobicity of the cell surface and drug susceptibility of Escherichia coli cultivated at high pressures up to 30 MPa

SummaryEscherichia coli was cultivated under hydrostatic pressures up to 30 MPa (300 bar) and then partitioned between an aqueous phase (physiological saline) and oil phase (n-hexadecane). The partition coefficients were used as measures of hydrophobicity of the surface of the cells and correlated with the susceptibility to an antimicrobial agent (dodecylpyridinium iodide). This agent is lethal to the cells and the effect of pressure on its concentration for a lethal effect on E. coli was determined. A good correlation was found between the hydrophobicity of the cells and their death rate on treatment with this reagent.

Enlargement of Grains of Silica Colloidal Crystals by Centrifugation in an Inverted-Triangle Internal-Shaped Container

We successfully fabricated large grains of silica colloidal crystals in an inverted-triangle internal-shaped container (inverted-triangle container) by centrifugation. The largest grain in the container was much larger than that in a container which has a flat bottom and constant width (flat-bottomed container). The edged bottom of the inverted-triangle container eliminated the number of the grains, and then the broadened shape of the container effectively widened the grains.

Gravitational Annealing of Colloidal Crystals

A silica colloidal crystal obtained by centrifugation at 9 G for 2 days in water was annealed by additional stronger centrifugation at 50 G for 5 days. The number of the striations observed in the colloidal crystal under crossed polarized light decreased at some parts in a growth container after the additional centrifugation, while the number also increased at the other parts. The decrease probably shows the shrinkage of the stacking disorders under high gravity, while the increase probably shows the production of new stacking disorders.