The overall objectives are to develop and apply state-of-the-art NMR techniques to the determination of the solution structural properties of complex biological macromolecules in order to elucidate their mechanism of action. The specific biological systems that will be studied by these methods are metallothioneins and a cyclosporin binding protein, termed cyclophilin. Metallothioneins (MTs): A variety of NMR methods will be used to fully resolve both the structural and metal-ion binding properties of this ubiquitous, metal-ion inducible, low molecular weight, cysteine-rich metal-binding protein isolated from various species in its long evolutionary history (fungus, yeast, invertebrate and mammalian). These very properties assures the ultimate importance of this protein in the cellular regulation of essential (Zn, Cu) and nonessential (Cd, Hg) metal ions. As a result, every effort will be made in these studies to elucidate those aspects of the structure of these different MTs which alter its metal-ion binding properties and to correlate this data with the presumed, but as yet undefined, function of this family of proteins. Cyclophilin is a ubiquitous, low molecular weight (17 Kd) cytosolic protein with high specificity for binding cyclosporin A, a potent immunosuppressant used clinically for the prevention of kidney, liver and heart allograft rejection. The specific objectives will be to use one and two-dimensional 1H NMR methods to elucidate the solution structural properties of cyclophilin, particularly as they relate to the binding site for cyclosporin. Studies such as these are of utmost importance to understanding the role of this protein in the pharmacologic activity of cyclosporin and its active metabolites, and the structure-activity relationships for cyclosporin. The long-term objectives would be to use NMR, in conjunction with other biochemical methods, to characterize the complex of cyclophilin with its intrinsic natural ligand when the latter is isolated. These studies would provide important new insight into the molecular mechanism of action of cyclosporin and the potential physiological significance of cyclophilin in the regulation of cellular metabolism.
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