The focus is on cysteine-rich proteins that form metal thiolate polymetallic clusters. A paradigm of this class is metallothionein (MT). Polymetallic clusters with distinct properties are induced by Zn (II) and Cu(I) ions. One goal is to determine the magnitude of structural reorganization in MT depending on the type of cluster formed. A second objective is to determine whether similar cluster structure alter the tertiary fold and function of these proteins. Three classes of molecules will be studied. First, a novel metallothionein implicated in Alzheimer's disease will be investigated. The MT, designated as GIF for Growth Inhibitory Factor, is active in inhibiting dendrite formation in neurons induced by Alzheimer's brain extracts. The tangled outgrowths of neurons that is characteristic of Alzheimer's disease may relate to the low concentration of GIF in Alzheimer's brain tissue. We propose experiments to determine which metallo-conformer of GIF is active in reversing the Alzheimer's extract induced proliferation of neurons. A series of experiments are proposed to map the segment of GIF responsible for activity. We plan to characterize the metal clusters in GIF to determine whether sequence differences in MT and GIF affect properties of the polymetallic clusters. The second class of proteins includes two fungal transcription factors. ACE and AMT1. Cu(I) binding to ACE! and AMT1 activates the factors for transcriptional activation of MT genes in Saccharomyces cerevisiae and Candida glabrata, respectively. We propose to characterize the Cu(I) thiolate polymetallic clusters in these two protein conformations. DNA binding sites of CuACE1 and CuAMT1 will be characterized with the goal of elucidating the structure of the transcriptionally active CuAMT1/DNA complex. The third class is the cysteine-rich sequence motif, designated LIM. The metal centers in two LIM-domain proteins, designated Cysteine-Rich Protein (CRP) and Cysteine- Rich Intestinal Protein (CRIP) will be studied to determine whether LIM proteins exhibit metal-induced conformational dynamics. A central postulate is that the structure and function of these classes of proteins are affected by the coordination chemistry of the metal centers. We eventually want to determine the role of specific metal ion binding in function. Molecules in these three classes exhibit a wide range of physiological functions from regulation of DNA transcription (ACE1 and AMT1), metal ion buffering (MT), inhibition of neuron outgrowth (GIF), protein-protein interaction (CRP) and perhaps metal transport (CRIP).
Showing the most recent 10 out of 83 publications
