Pressure unfolding studies

The early pressure unfolding studies cited above revealed all of the essential parameters for describing combined temperature-pressure effects. Hawley first demonstrated that protein unfolding p-T diagrams were elliptical in shape... 

We determined several years ago the temperature dependence of the pressure unfolding of Snase [14] using fluorescence, FTIR and SAXS to build the p-T phase diagram (Fig. 9.3). These studies showed a clear decrease in the absolute value of the volume change for unfolding as a function of temperature, although the uncertainty in the recovered values of AV did not allow us to conclude unequivocally in a linear dependence. Nonetheless, in the absence of any further information we assumed linearity and hence calculated from the slope the change in thermal expansivity between the folded and the unfolded state to be on the order of 1 ml mol-1 K-1. 

ZhangJ, Peng X, Jonas A, andjonasj. NMR Study of the Cold, Heat and Pressure Unfolding of Ribonuclease A. Biochemistry 1995 34 8631-8641. 

Doster, W., and Gebhardt, R. (2003) High pressure-unfolding of myoglobin studied by dynamic neutron scattering, Chem. Phys. 292, 383-387... 

Pressure-jump studies were carried out to characterize the kinetics of the folding-unfolding processes in proteins with a whole number of different spectroscopic methods (for review see 107 and 108). In case of the small protein PI3MTCP1 gjjjg report a real-time observation of pressure-... 

Pressure induced unfolding studies are more rarely performed since a specialized high pressure cell is required. 

Directed evolution as a tool to probe the basis of protein structure, stability, and function is in its infancy, and many fruitful avenues of research remain to be explored. Studies so far have focused on proteins that unfold irreversibly, making detailed thermodynamic analysis impossible. The application of these methods to reversibly folding proteins could provide a wealth of information on the thermodynamic basis of high temperature stability. A small number of studies on natural thermophilic proteins have identified various thermodynamic strategies for stabilization. Laboratory evolution makes it possible to ask, for example, whether proteins have adopted these different strategies by chance, or whether certain protein architectures favor specific thermodynamic mechanisms. It will also be possible to determine how other selective pressures, such as the requirement for efficient low temperature activity, influence stabilization mechanisms. The combination of directed evolu-... 

We can reasonably assume two major contributions to the difference in specific volume between the unfolded and folded states of a protein. The first contribution is that arising from the decrease in solvent-excluded volume when the tightly, but of course not perfectly, packed protein folded structure is disrupted. Water molecules enter this volume, thereby decreasing the overall volume of the protein solvent system. The magnitude of this contribution is a specific property of the protein, both in its folded and unfolded state. The second contribution arises from the change in the volume of the water molecules that hydrate the newly exposed protein surface area, relative to their volume in the bulk. Much of our present understanding of the contribution of differential hydration volume has come from recent studies of model compounds and proteins based on PPC. This technique, developed by Brandts and coworkers [17] and recently reviewed by us [16,18], is based on the measurement of the heat released or absorbed upon small (e.g., 0.5 MPa) pressure... 

TAS = 0) obtained with the 5 10°C temperature shift and the proportionally small optical changes in UV-Visible spectroscopy associated with the transient disequilibrium state. However, these limitations do not seem to apply to protein folding studies in which changes of tryptophan fluorescence or that of the fluorescently labeled proteins were monitored. In addition to a temperature jump, protein unfolding can also be rapidly initiated by a pressure jump. The dead time of the recently developed pressure-jump instrument is 50 ts for a pressure jump of 100 bar. ... 

Much of the solution conformational work was done in 1960—1972, when CD, ORD, and Raman spectroscopic apparatus were not very satisfactory. Johnson (1988) indicated the scope of secondary structural investigation of proteins with modern CD apparatus. With the increased accessibility of the UV region, such measurements need to be done on a-lactalbumin and lysozyme in a variety of environments over a wide temperature range. Kauzmann (1987) indicated that, in considering the thermodynamics of the unfolding of proteins, we are tending to avoid the hard experiments. Thus, spectroscopic studies of the effects of high pressure on these proteins are sorely needed. 

The molar volume change AV° is small for most reactions in solution, which requires large pressure changes (>50 atm) for a measurable perturbation of an equilibrium [30], This technique has not found much use in electron-transfer reactions, but various absorption-desorption phenomena, association-dissociation equilibria, and structural relaxations have been studied successfully. Pressure jump is also quite useful in biological studies of folding and unfolding of proteins and DNA owing to the pressure sensitivity of these processes [31],... 

There are several reasons to measure the effect of pressure on a wide variety of thermodynamic systems. Perhaps the most important argument is that one can separate the effects of volume and thermal energy changes, which appear simultaneously in temperature experiments . Moreover, high pressure can induce unfolding of protein in a different way from thermal denaturation. The pressure studies have considerably increased in the last decades" . 

The stage is now well set for further work addressing more complex questions, such as the study of the folding reaction of oligomers and protein complex formation as well as for studies of aggregation phenomena. Only a few studies have been performed in this direction so far [9-11, 18, 114, 124]. At pressures of 4-8 kbar, most small monomeric proteins unfold... 

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