Ion channels under high pressure

Hydrostatic pressure (<100 MPa) affects the kinetics of ion channels but not their conductance. In voltage-gated channels, pressure acts on the movement of the charge sensor and on the conformational change involved in opening the channel pore. It has also been shown to act on N-type inactivation ball-binding, C-type inactivation and to activate BK channels. There is little doubt that these are sites of adaptation to high pressure in the channels of deep-sea animals. Pressure studies should not be regarded in isolation; they relate well to experiments using other variables such as osmotic pressure, solvent viscosity and temperature. Furthermore ion channels could transduce pressure in the sensory system of aquatic animals, providing information about the animals' depth, a prediction supported by our knowledge of heat-activated channels in mammals.     

UV-visible derivative spectroscopy under high pressure

High hydrostatic pressure affects proteins, changing their intra- or intermolecular interactions, conformation and solvation. How to detect these changes? In this paper, via some selected examples, we show the potentiality (but also the limits) of the ultraviolet derivative spectroscopy specially adapted to high pressure experiments.     

Starch gelatinization under pressure studied by high pressure DSC

The gelatinization process of waxy corn starch under different pressures up to 10.0 MPa was investigated using a high pressure DSC. Compressed air and carbon dioxide were used as pressure resources. Effect of pressure and annealing under pressure on gelatinization of waxy corn starch was systematically studied, in particular on the gelatinization temperature and enthalpy. The results show that the peak temperature of gelatinization was increased slightly initially then remained stable with increasing pressure. The gelatinization enthalpy was decreased under pressure processing. Annealing the starch under pressure condition, just below its gelatinization temperature, increased gelatinization temperature but kept gelatinized enthalpy constant. Morphologies of starch granules treated under pressure were studied using an optical microscope and SEM. There is no discernable difference of starch granules treated with and without pressure, which indicates the pressures are not high enough to destroy crystalline structure. The intensity of the pressure acts as a key factor to influence the gelatinization of starch rather than the nature of the gas. Effect of pressure on the multi-endotherm detected by DSC for starch with intermediate water is used to study the mechanisms. The effect of pressure can be explained by the enhancement of water diffusion in the amorphous range.     

Cold denaturation of proteins under high pressure

The advantageous usage of the high pressure technique in studies of cold denaturation of proteins is reviewed, with a brief explanation of the theoretical background of this universal phenomenon. Various experimental results are presented and discussed, explaining the plausible image of the cold denatured state of proteins. In order to understand more clearly this phenomenon and protein structure transition in general, several studies on model polymer systems are also reviewed.     

Protein crystallization in microgravity.

A space experiment involving protein crystallization was conducted in a microgravity environment using the space shuttle "Endeavour" of STS-47, on a 9-day mission from September 12th to 20th in 1992. The crystallization was carried out according to a batch method, and 5 proteins were selected as flight samples for crystallization. Two of these proteins: hen egg-white lysozyme and co-amino acid: pyruvate aminotransferase from Pseudomonas sp. F-126, were obtained as single crystals of good diffraction quality. Since 1992 we have carried out several space experiments for protein crystallization aboard space shuttles and the space station MIR. Our experimental results obtained mainly from hen egg-white lysozyme are described below, focusing on the effects of microgravity on protein crystal growth.     

Protein crystallization and phase diagrams

The phase diagram is a map which represents the state of a material (e.g., solid and liquid) as a function of the ambient conditions (e.g., temperature and concentration). It is therefore a useful tool in processing many different classes of materials. In this article, methods to determine the phase diagram of an aqueous solution of a globular protein are described, focusing on the solid (crystal) and condensed liquid states. The use of the information contained in the phase diagram for protein crystallization is also discussed.     

Stability of subtilisin and lysozyme under high hydrostatic pressure

The stabilities of subtilisin and lysozyme under hydrostatic pressures up to 200 MPa were investigated for up to 7 days at 25 degrees C. Methods were chosen to assess changes in tertiary and secondary protein structure as well as aggregation state. Tertiary structure was monitored in situ with second derivative UV spectroscopy and after pressure treatment by dynamic light scattering and second derivative UV spectroscopy. Secondary structure and potential secondary structural changes were characterized by second derivative FTIR spectroscopy. Changes in aggregation state were assessed using dynamic light scattering. Additionally, protein concentration balances were carried out to detect any loss of protein as a function of pressure. For the conditions tested, neither protein shows measurable changes in tertiary or secondary structure or signs of aggregation. Lysozyme concentration balances show no dependence on pressure. Subtilisin concentration balances at high protein concentration (4 mg/mL and higher) do not show pressure dependence. However, the concentration balances carried out at 0.4 mg/mL show a clear sign of pressure dependence. These results may be explained by protein interaction with the vial surface and appear to be rate limited by the equilibrium between active and inactive protein on the surface. Pressure increases protein loss, and the estimated partial molar volume change between the two states is estimated to be -20 +/- 10 mL/mol.     

Bilayer phase transitions of N-methylated dioleoylphosphatidylethanolamines under high pressure

The bilayer phase transitions of four kinds of unsaturated phospholipids with different-sized polar head groups, dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidyl-N-methylethanolamine (DOMePE), dioleoylphosphatidyl-N,N-dimethylethanolamine (DOMe2PE) and dioleoylphosphatidylcholine (DOPC), were observed by means of differential scanning calorimetry (DSC) and high-pressure light-transmittance. DSC thermogram and light-transmittance curve for each phospholipid vesicle solution exhibited only one phase transition under ambient pressure, respectively. The light-transmittance of DOPC solution at pressure higher than 234 MPa abruptly increased stepwise at two temperatures, which corresponds to the appearance of stable subgel and lamellar gel phases under high pressure in addition to the liquid crystal phase. The constructed temperature (T)-pressure (p) phase diagrams were compared among these phospholipids. The phase-transition temperatures of the phospholipids decreased stepwise by N-methylation of the head group. The slops of the T-p phase boundary (dT/dp) of DOPE, DOMePE and DOMe2PE bilayers (0.127-0.145 K MPa-1) were found to be close to that of the transition from the lamellar crystal (or subgel; Lc) phase to the liquid crystal (Lalpha) phase for DOPC bilayer (0.131 K MPa-1). On the other hand, the dT/dp value of the main transition from the lamellar gel (Lbeta) phase to the Lalpha phase for DOPC bilayer (0.233 K MPa-1) was significantly different from that of the Lc/Lalpha transition, hence both curves intersected with each other at 234 MPa. The thermodynamic quantities associated with the phase transition of DOPE, DOMePE and DOMe2PE bilayers had also similar values to those for the Lc/Lalpha transition of DOPC bilayer. Taking into account of the values of transition temperature, dT/dp and thermodynamic quantities compared with the corresponding results of saturated phospholipids, we identified the phase transitions observed in the DOPE, DOMePE and DOMe2PE bilayers as the transition from the Lc phase to the Lalpha phase although they have been regarded as the main transition in the previous studies. The Lbeta phase is probably unstable for DOPE, DOMePE and DOMe2PE bilayers at all pressures, it exists as a metastable phase at pressures below 234 MPa while as a stable phase at pressures above 234 MPa in DOPC bilayer. The difference in phase stability among the phospholipid bilayers is originated from that in hydration structure of the polar head groups.     

Structural stability of human butyrylcholinesterase under high hydrostatic pressure

Human butyrylcholinesterase is a nonspecific enzyme of clinical, pharmacological and toxicological significance. Although the enzyme is relatively stable, its activity is affected by numerous factors, including pressure. In this work, hydrostatic pressure dependence of the intrinsic tryptophan fluorescence in native and salted human butyrylcholinesterase was studied up to the maximum pressure at ambient temperature of about 1200 MPa. A correlated large shift toward long wavelengths and broadening observed at pressures between 200 and 700 MPa was interpreted as due to high pressure-induced denaturation of the protein, leading to an enhanced exposure of tryptophan residues into polar solvent environment. This transient process in native butyrylcholinesterase presumably involves conformational changes of the enzyme at both tertiary and secondary structure levels. Pressure-induced mixing of emitting local indole electronic transitions with quenching charge transfer states likely describes the accompanying fluorescence quenching that reveals different course from spectral changes. All the pressure-induced changes turned irreversible after passing a mid-point pressure of about 400 ± 50 MPa. Addition of either 0.1 M ammonium sulphate (a kosmotropic salt) or 0.1 M lithium thiocyanate (a chaotropic salt) to native enzyme similarly destabilized its structure.     

beta-lactoglobulin under high pressure studied by small-angle neutron scattering

We used small-angle neutron scattering to study the effects of the high hydrostatic pressure on the structure of beta-lactoglobulin. Experiments were carried out at pH 7 on the dimeric form of the protein in a pressure range going from 50 MPa to 300 MPa. These measurements allow the protein size and the interactions between macromolecules to be studied during the application of pressure. Increasing pressure up to 150 MPa leads to a swollen state of the protein that gives rise to an increase of the radius of gyration by about 7%. Within this pressure range, we also show that the interaction between macromolecules weakens although it remains repulsive. The measurements show an aggregation process occurring above 150 MPa. From the spectra analysis, it appears that the aggregation occurs mainly by association of the dimeric units.     

Protein crystallization: virtual screening and optimization

Advances in genomics have yielded entire genetic sequences for a variety of prokaryotic and eukaryotic organisms. This accumulating information has escalated the demands for three-dimensional protein structure determinations. As a result, high-throughput structural genomics has become a major international research focus. This effort has already led to several significant improvements in X-ray crystallographic and nuclear magnetic resonance methodologies. Crystallography is currently the major contributor to three-dimensional protein structure information. However, the production of soluble, purified protein and diffraction-quality crystals are clearly the major roadblocks preventing the realization of high-throughput structure determination.

This paper discusses a novel approach that may improve the efficiency and success rate for protein crystallization. An automated nanodispensing system is used to rapidly prepare crystallization conditions using minimal sample. Proteins are subjected to an incomplete factorial screen (balanced parameter screen), thereby efficiently searching the entire “crystallization space” for suitable conditions. The screen conditions and scored experimental results are subsequently analyzed using a neural network algorithm to predict new conditions likely to yield improved crystals. Results based on a small number of proteins suggest that the combination of a balanced incomplete factorial screen and neural network analysis may provide an efficient method for producing diffraction-quality protein crystals.     

Protein crystallization in the structural genomics era

There are five broad areas where noteworthy advances have occurred in the field of macromolecular crystallization in the past 10 years, though some areas have seen the major part of those advances in only the last two years. This is largely a consequence of the international structural genomics initiative and its early results. The five areas are: (1) Physical studies and characterization of the protein crystallization process; (2) Development of new practical approaches and procedures; (3) The implementation of protein engineering by genetic means to enhance both purification and crystallization; (4) The creation of new screening conditions based on information and databases emerging from structural genomics; and (5) Development and implementation of automation, robotics, and mass screening of crystallization conditions using very small amounts of protein. A brief summary is provided here of the progress in the past few years and the influence of the structural genomics project.     

Protein under pressure: molecular dynamics simulation of the arc repressor

Experimental nuclear magnetic resonance results for the Arc Repressor have shown that this dimeric protein dissociates into a molten globule at high pressure. This structural change is accompanied by a modification of the hydrogen-bonding pattern of the intermolecular beta-sheet: it changes its character from intermolecular to intramolecular with respect to the two monomers. Molecular dynamics simulations of the Arc Repressor, as a monomer and a dimer, at elevated pressure have been performed with the aim to study this hypothesis and to identify the major structural and dynamical changes of the protein under such conditions. The monomer appears less stable than the dimer. However, the complete dissociation has not been seen because of the long timescale needed to observe this phenomenon. In fact, the protein structure altered very little when increasing the pressure. It became slightly compressed and the dynamics of the side-chains and the unfolding process slowed down. Increasing both, temperature and pressure, a tendency of conversion of intermolecular into intramolecular hydrogen bonds in the beta-sheet region has been detected, supporting the mentioned hypothesis. Also, the onset of denaturation of the separated chains was observed.