The effects of pressure on cultures of Lactobacillus plantarum were characterized by determination of the viability and activity of HorA, an ATP-binding cassette multidrug resistance transporter. Changes in the membrane composition of L. plantarum induced by different growth temperatures were determined. Furthermore, the effect of the growth temperature of a culture on pressure inactivation at 200 MPa was determined. Cells were characterized by plate counts on selective and nonselective agar after pressure treatment, and HorA activity was measured by ethidium bromide efflux. Fourier transform-infrared spectroscopy and Laurdan fluorescence spectroscopy provided information about the thermodynamic phase state of the cytoplasmic membrane during pressure treatment.

Temperature- and pressure-induced denaturation of the protein ubiquitin was investigated using FT-IR spectroscopy. On the basis of IR spectral parameters, different states are distinguished and a pressure/temperature-stability diagram of the protein has been determined. The evolution of the secondary structures with temperature illustrates that the band intensities of disordered structures decrease at the expense of the formation of intermolecular beta-sheets at 83 degrees C, pD 7, and ambient pressure, with the population of intramolecular beta-sheets and alpha-helices remaining essentially unchanged. At ambient temperature (T = 21 degrees C) and pD 7, ubiquitin denatures at 5.4 kbar. Contrary to other proteins studied so far, features of secondary structure of ubiquitin remain distinct at high pressure, suggesting that part of this small protein rearranges and does not unfold to disordered structures. The secondary structural changes during compression and decompression are fully reversible, and no aggregation occurs. With corresponding measurements of the pressure-induced denaturation of ubiquitin at different temperatures, a p/T-stability diagram of ubiquitin could be obtained. Furthermore, kinetic FT-IR measurements were carried out using the pressure-jump relaxation technique. The denaturation process is shown to occur on a time scale which is about twice as long as that of the renaturation process, and both processes are much slower than the unfolding-refolding kinetics observed at ambient pressure.

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FT-IR spectroscopy was used to study the effects of various chaotropic and kosmotropic cosolvents (glycerol, sucrose, sorbitol, K(2)SO(4), CaCl(2), and urea) on the secondary structure and thermodynamic properties upon unfolding and denaturation of staphylococcal nuclease (Snase). The data show that the different cosolvents have a profound effect on the denaturation pressure and the Gibbs free energy (DeltaG(o)) and volume (DeltaV(o) change of unfolding. Moreover, by analysis of the amide I' infrared bands, conformational changes of the protein upon unfolding in the different cosolvents have been determined. An increase, a reduction, or an independence of the volume change of unfolding is observed, depending on the type of cosolvent, which can at least in part be attributed to the formation of a different unfolded state structure of the protein. The data are compared with the corresponding thermodynamic values of DeltaV(o) for the temperature-induced unfolding process of Snase as obtained by pressure perturbation calorimetry, and significant differences are observed and discussed.

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