Many of the large proteins found in plasma and th extracellular matrix (ECM) share a number of general featrues. Fibronectin, tenascin, laminin, fibrinogen, von Willebrand factor, and thrombospondin, for example, are all comprised of repeated homologous subunits and appear in electron micrographs as long flexible molecules that are often characterized as """"""""beads on a string."""""""" These subunits are frequently members of larger protein families, and domains homologous to these subunits are found in numerous proteins. Specific interactions and binding sites have been associated with individual subdomains, and interactions with multiple extracellular proteins, proteoglycans, and cell-surface receptors have been demonstrated. Through these multiple interactions the molecular meshwork of thrombi and the extracellular compartments of tissues are formed. The ability of small peptide fragments from individual subdomains to block interactions with other molecules and thus inhibit functions of the whole protein has led to the investigation of peptide analogues as potential drugs. The best characterized of these inhibitory peptides are those that contain the Arg-Gly-Asp (RGD) sequence. Several proteins contain domains that use this sequence to mediate interactions with members of the integrin family of cell-surface receptors, and these interactions are involved in such physiological processes as thrombosis, inflammation, and osteoclast- mediated bone resorption. I plan to pursue x-ray diffraction studies of subdomain and multi-subdomain fragments of large extracellular proteins. When possible, co-crystals of these domains with ligands or receptors will also be studied. I have produced diffraction-quality crystals of a fragment of human fibronectin containing the seventh through the RGD-containing tenth type III repeats. I propose to: (1) solve and refine the crystal structure of this fragment of fibronectin, (2) use these and other available relevant structures to determine general features of the structure and interdomain interactions of type III repeats, (3) undertake the expression and crystallographic analysis of A and C domains from von Willebrand factor, including the RGD- containing C1 domain, and (4) attempt to produce co-crystals of these domains and potential ligands for crystallographic analysis.
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