The long-term objective of the proposed research is to contribute to the understanding of the stability, folding intermediates, and dynamics of proteins by using pressure as the fundamental thermodynamic variable. Unique high pressure equipment, suitable for measurements from 0.1 MPa (l bar) to l GPa (10 kbar) and from -25 degrees C to lOO degrees C, will be used in conjunction with a variety of l-D and 2-D high resolution NMR techniques to study the effects of pressure on the unfolding/folding equilibria, conformational states of native and compact denatured states, and the dynamic properties of several model proteins. In addition, the cold-denaturation behavior of these proteins will be studied by taking advantage of the depression of the freezing point of water by pressure. The pressure, cold, heat, and chemically denatured states of the model proteins will be examined and compared, with special emphasis on the compact denatured states observed by cold and pressure denaturation. The model proteins selected for these studies include hen egg-white lysozyme, bacteriophage T4 lysozyme, horse myoglobin, sperm-whale myoglobin, staphylococcal nuclease, and bovine pancreatic ribonuclease A, as well as selected mutants of T4 lysozyme, sperm-whale myoglobin, staphylococcal nuclease, and bovine pancreatic ribonuclease A. To complement the NMR measurements at high pressure, especially on the compact denatured states of the model proteins and their mutants, circular dichroism (CD) and laser-photo chemically induced dynamic nuclear polarization (CIDNP) experiments will be performed. High-pressure CD cells and CIDNP probes will be designed and constructed.
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