Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology最新文献

The pressure–temperature phase diagram of various biomolecules is reviewed. Special attention is focused on the elliptic phase diagram of proteins. The phenomenological thermodynamic theory describing this diagram explains the heat, cold and pressure denaturations in a unified picture. The limitations and possible developments of this theory are discussed as well. It is pointed out that a more complex diagram can be obtained when the intermolecular interactions are also taken into account. In this case metastable states appear on the pressure–temperature (p–T) diagram due to intermolecular interactions. Pressure–temperature phase diagrams of other biopolymers are also discussed. While the p–T diagrams of helix–coil transition of nucleic acids and of gel–liquid crystal transition of lipid bilayers are non-elliptical, those of gelatinization of starch and of phase separation of some synthetic polymers show an elliptic profile, similar to that of proteins. Finally, the p–T diagram of bacterial inactivation is shown to be elliptic. From the point of view of basic science, this fact shows that the key factor of inactivation should be the protein type, and from the viewpoint of practical applications, it serves as the theoretical basis of pressure treatment of biosystems.

A fundamental factors, pressure (P), is indispensable to develop and support applications in the field of bioscience and biotechnology. This short sentence describes an example how high pressure bioscience and biotechnology, which started from applied science, stimulates challenges of basic science and pure science in the biology-related fields including not only food science, medicine, and pharmacology but also biochemistry, molecular biology, cell biology, physical chemistry, and angineering.

After a brief introduction of the potentialities of Trp phosphorescence spectroscopy for probing the conformation and flexibility of protein structure, this presentation summarizes the effects of hydrostatic pressure (up to 3 kbar) on the native fold of monomeric and oligomeric proteins as inferred from the variation of the intrinsic phosphorescence lifetime and the oxygen and acrylamide bimolecular quenching rate constants of buried Trp residues. The pressure/temperature response of the globular fold and modulation of its dynamical structure is analyzed both in terms of a reduction of internal cavities and of hydration of the polypeptide. The implications of these findings for the thermodynamic stability of proteins and for the determination of subunit dissociation equilibria under high pressure conditions are also discussed.

We review the results of compressibility studies on proteins and low molecular weight compounds that model the hydration properties of these biopolymers. In particular, we present an analysis of compressibility changes accompanying conformational transitions of globular proteins. This analysis, in conjunction with experimental compressibility data on protein transitions, were used to define the changes in the hydration properties and intrinsic packing associated with native-to-molten globule, native-to-partially unfolded, and native-to-fully unfolded transitions of globular proteins. In addition, we discuss the molecular origins of predominantly positive changes in compressibility observed for pressure-induced denaturation transitions of globular proteins. Throughout this review, we emphasize the importance of compressibility data for characterizing protein transitions, while also describing how such data can be interpreted to gain insight into role that hydration and intrinsic packing play in modulating the stability of and recognition between proteins and other biologically important compounds.

A method was developed to investigate the stability of protein–nucleic acid complexes using hydrostatic pressure during electrophoretic gel mobility shift analysis. The initial system probed by this technique was the well-characterized cognate BamHI–DNA complex. Band shift analysis at several elevated pressures found the equilibrium dissociation (K_d) constant to be dependent on pressure, which allowed the volume change of dissociation (ΔV) to be calculated. In order to describe the effects of pressure on the specific BamHI–DNA complex at the molecular level, molecular dynamics simulations at both ambient and elevated pressure was performed. Comparison of the simulation trajectories identified several individual BamHI–DNA contacts that are disrupted due to pressure. The disruption of these contacts can be attributed to an observed pressure-induced increase in hydration at the protein–DNA interface during the elevated pressure simulation.

The combination of advanced high-resolution nuclear magnetic resonance (NMR) techniques with high-pressure capability represents a powerful experimental tool in studies of protein folding. This review is organized as follows: after a general introduction of high-pressure, high-resolution NMR spectroscopy of proteins, the experimental part deals with instrumentation. The main section of the review is devoted to NMR studies of reversible pressure unfolding of proteins with special emphasis on pressure-assisted cold denaturation and the detection of folding intermediates. Recent studies investigating local perturbations in proteins and the experiments following the effects of point mutations on pressure stability of proteins are also discussed. Ribonuclease A, lysozyme, ubiquitin, apomyoglobin, α-lactalbumin and troponin C were the model proteins investigated.

Ion channels are distinctive membrane proteins which provide a gated pathway for diffusing ions. High pressure (<100 MPa) affects the kinetics of gating but not the conductance of the channel. Dynamic structural studies of channels at high pressure are, thus far, conspicuously absent but functional properties are studied at the single channel level with the patch clamp technique.

Pressures between 10 and 100 MPa can exert powerful effects on the growth and viability of organisms. Here I describe the effects of elevated pressure in this range on mesophilic (atmospheric pressure adapted) and piezophilic (high-pressure adapted) microorganisms. Examination of pressure effects on mesophiles makes use of this unique physical parameter to aid in the characterization of fundamental cellular processes, while in the case of piezophiles it provides information on the essence of the adaptation of life to high-pressure environments, which comprise the bulk of our biosphere. Research is presented on the isolation of pressure-resistant mutants, high-pressure regulation of gene expression, the role of membrane lipids and proteins in determining growth ability at high pressure, pressure effects on DNA replication and topology as well as on cell division, and the role of extrinsic factors in modulating enzyme activity at high pressure.