ESR SPIN LABEL STUDIES OF FIBRONECTINS

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Fibronectin is a major protein of blood and tissues that plays a central role in cell adhesion. The goal of GM35719 is to gain a deeper understanding of the three-dimensional relationship of various regions of fibronectin, and of the structural basis for ligand binding and molecular expansion. The specific aim 1 of this renewal application is to characterize the dynamic interactions between various functional domains of plasma fibronectin using electron spin resonance (ESR) spectroscopy. The motions of the fibronectin molecule and its fragments under various conditions will be studied by estimating the effective rotational correlation times from the experimental spectra using computer simulations. The specific aim 2 is to determine the intramolecular distances of the fibronectin molecule using fluorescence energy transfer techniques. These distances will be used as new constraints to refine the current three-dimensional model of fibronectin structure. The specific aim 3 is to study the non-covalent association of fibronectin dimers by using monomeric fibronectin. The contact sites and the nature of non-covalent association between the two monomers of fibronectin, which lack the interchain disulfide bonds, will be studied by using both ESR and fluorescence spectroscopy. The specific aim 4 is to characterize recombinant fibronectin fragments expressed in bacterial systems using molecular cloning techniques. Site-directed mutagenesis by substitution of a cysteine residue involved in interchain disulfide bonding with a serine residue will be employed to determine whether the two interchain disulfide bridges in the carboxyl ends of the molecule are arranged in parallel or antiparallel fashion. The specific aim 5 is to investigate the structure and dynamics of the recombinant fibronectin fragments using a combination of site-directed mutagenesis and biophysical techniques. Fibronectin fragments will be genetically engineered to contain single selective labeling sites, one at a time, without affecting the protein function. This permits the placement of spin labels or fluorescent labels into the recombinant fibronectin fragment and the elucidation of detailed structural information through ESR or fluorescence analysis. The proposed studies will provide previously unavailable information regarding the three-dimensional structure of the fibronectin molecule and mechanisms by which functional unmasking of certain domains occurs in response to ligand-induced conformational alterations. This information should provide a basis for explaining important roles of fibronectin in cell attachment and migration, embryogenesis, wound healing, and oncogenic transformation.