HIGH PRESSURE NMR STUDIES OF PROTEINS AND MEMBRANES

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The long-term objective of the proposed research is the elucidation of the effects of pressure on the dynamics and structure of proteins and biological membranes. Pressure is a fundamental thermodynamic variable that provides information on processes that involve changes in volume, including the folding and unfolding of proteins, lipid phase changes in membranes, and the modulation of the fluidity and interfacial properties of membranes. The proposed work focuses on the effects of pressure on the dynamics, phase state, and conformation of several model systems: homopolypeptides (poly-L-lysine, poly-L-glutamic acid, and poly-N -(3- hydroxypropyl)-L-glutamine), hen egg-white lysozyme, sperm whale myoglobin, and various pure phospholipids in the form of sonicated vesicles or multilamellar dispersions. A variety of advanced high resolution NMR methods, using H, H, P nuclei, will be employed together with unique high-pressure NMR probes, in the range from 1 bar to 1O kbar and from 0 to, 100 degree C. In the proteins and the homopolypeptides, selected 1H and 13C resonances will be used to study the dynamics in various regions of the macromolecules, and to examine the thermodynamics and kinetics of the folding-unfolding transitions. The dynamics of phospholipid molecules in liposomes will be studied by 13C NMR: the order and con-formation of the acyl chains and the head groups will be investigated by 13C-NMR, using deuterated lipids, and by H-2D NMR techniques: and phase changes will be examined by 31P and H NMR methods. In addition, slow motions and the lateral diffusion of phospholipids in liposomes will be investigated by rotating frame NMR relaxation techniques.