Fibronectin (FN) forms the primordial extracellular matrix in embryonic development and wound healing. Remarkably, the assembly of FN matrix fibrils is one of the least understood macromolecular assemblies. Our major goal in the next five years is to determine the nature of the intermolecular bonds that form the fibrils. We propose that there are two steps in fibril assembly. The first step, already understood qualitatively, is association of molecules by reversible bonds, primarily involving the N-terminal 70k fragment. The second step, still completely unknown, is formation of strong, irreversible bonds. We propose to study the first step quantitatively, using Biacore and analytical centrifugation to determine the stoichiometry and Kd of the reversible bonds. We then propose a novel hypothesis for the second step - that the strong bonds are formed by a domain swapping mechanism. Specifically we propose that FN-III domains open up and swap beta strands with domains on another molecule. We propose to test this by engineering """"""""disulfide locks"""""""" in the suspected FN-III domains, and testing if this blocks the ability to form fibrils. We have recently engineered a disulfide lock that inhibited assembly of """"""""super fibronectin,"""""""" and are now ready to test this for assembly of recombinant FN fragments and matrix fibrils in cell culture. We also propose x-ray crystallography of FN-III domains 1-2, which are key players in assembly of sFN and matrix fibrils. We have shown that FN matrix fibrils are very elastic, and are mostly stretched 2-5 times rest length. A second project is to investigate the mechanism of elasticity, and to distinguish between two proposed mechanisms. One mechanism proposes that FN-III domains unfold upon stretching, and the other that stretching is achieved by a compact-to-extended conformation change of the whole molecule, without domain unfolding. We propose to design intramolecular force sensors, which will tell us whether the force is sufficient to unfold FN-III domains. The sensors will be based on FRET, with CFP and YFP separated by entropic springs of different strength. After our initial applications and testing in FN, these sensors should be widely applicable to determine the tension in other ECM and cytoskeletal systems.
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