MULTINUCLEAR NMR PROBES OF BIOLOGICAL SYSTEMS

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The long-term objectives are to develop and apply state-of-the-art NMR techniques to the determination of the solution structural properties of a ubiquitously distributed protein involved in metal homeostasis, metallothioneins, MTs. This information is essential for the elucidation of the molecular mechanism of action of MT in metal regulation. An integral component of the proposed research is to contribute the adaptation/refinement of novel new NMR methods for macromolecular studies. While detailed structural information has recently become available through NMR and X-ray crystallographic methods for the Cd, Zn containing mammalian MT, similar structural information is not available for the Cu+ containing forms of the mammalian MT, nor for the MTs from other species (Saccharomyces cerevisiae, Neurospora crassa). Specific NMR experiments are proposed using the Ag+ ion as a chemically and magnetically (spin I=1/2) isomorphic metal ion substitute for Cu+ for the determination of the solution structure of this metal form of MT. NMR methods will also be used for the full solution structure determination of MTs from the marine crustaceans, the American lobster and the blue crab, which bind the divalent metal ions, Cd and Zn, and/or the monovalent metal ion, Cu. Selected MT samples from these sources, which will include recombinant forms of Chinese hamster MT, will concurrently and/or subsequently be used in experiments whose objectives are to delineate the kinetics and mechanistic features associated with this protein's physiological role in metal homeostasis. It is tempting to speculate that because of the analogies of the metal- binding motifs in both the transcription factors and MT, that the latter could have a metalloregulatory function in cellular repair processes, growth, and differentiation.