Metalloprotein design requires a wide spectrum of knowledge, originating with an understanding of the structural and chemical basis for metal ion affinity, specificity and function. This project is designed to attain such knowledge for one of the primary classes of metalloprotein, those whose activity or intermolecular interactions are modulated by conformational changes induced by ion binding. More specifically, the solution structure and dynamics of a novel zinc and calcium binding protein, calcyclin, will be examined using nuclear magnetic resonance spectroscopy. Calcyclin undergoes growth-related changes of expression, being mainly expressed in G1 phase. It has been implicated in the regulation of cell growth and cell proliferation, is abundant in certain human breast cancer cell lines, and is overexpressed in human myeloid leukemias and human melanoma cell lines. A growing body of evidence points to a specific regulatory function for this protein involving calcium-dependent modulation of the activity of a membrane bound annexin, CAP-50. Calcyclin exhibits high affinity binding sites for one zinc and two calcium ions. An analysis of its amino acid sequence strongly suggests that the zinc binding site is formed as a byproduct of the details of the protein fold and hence, that the metal binding sites overlap. This raises the distinct possibility of calcium signaling being modulated by zinc binding, which would correspond to a novel regulatory mechanism for the calmodulin superfamily. The specific purpose of this project is to determine the molecular organization of the protein, and to probe the nature of and relationships between the zinc and calcium binding sites.
Three specific aims are proposed: (1) determine the three-dimensional solution structure of calcyclin from rabbit lung with the zinc and calcium sites empty, by multidimensional 1H NMR spectroscopy; (2) determine the structure of the protein in the absence of calcium with zinc present, in the absence of zinc but with calcium present, and with all three metal sites occupied, by multi-dimensional 1H and heteronuclear NMR approaches; (3) probe the factors governing the interactions between the zinc and calcium binding sites by preparing designed site-directed mutants of calcyclin and analyzing their structure by NMR. Beyond basic structural information, this project will contribute to the breadth and diversity of the Program by providing information on a unique example of site-site interactions in a mixed metal system, an areas for which there is only a limited amount of structural information currently available.
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