PROJECT SUMMARY/ABSTRACT Collagens are essential components of the extra-cellular matrix and basement membranes, where they serve to bolster tissue integrity, mediate cell migration, and organize signaling. Collagen proteins have three domains: the C-terminal trimerization domain (CTD/NC1), the collagen domain and the 7S N-terminal domain. Collagens undergo folding followed by trimerization and collagen triple helix formation in the ER prior to secretion to the extra-cellular environment. Trimerization of the NC1 domain is the first step in collagen folding and is required for formation of the collagen triple helix. Mutations in collagens result in severe diseases, such as Osteogenesis Imperfecta, Alport’s Disease and Cerebral Small Vessel Disease which impair the integrity of bones, kidneys and blood vessels in the brain and eye respectively. Although many disease-causing mutations in collagens have been identified, there still is an incomplete understanding of the mechanisms of pathogenesis. The goal of this proposal is to understand how perturbing the initial trimerization effects the maturation and biosynthesis of collagen IV. In my first aim, I will develop new cell-based assays to quantify the effect of NC1 domain perturbation on collagen assembly. Specifically, I have developed a split luciferase assay that measures NC1 domain interactions in isolation and have begun to quantify the effect of pathogenic mutations. Additionally, I will develop complementary cell lines that express full-length collagen IV, including the most interesting mutants identified in the split luciferase assays, to better understand the functional consequences of these perturbations. In my second aim, I have designed point mutations at the NC1 interface, and identified those that increase or decrease trimer stability. Excitingly, one of these mutations significantly increased NC1 association and secretion. Together, these studies will reveal the mechanisms of collagen-associated disease and suggest ways of circumventing them. The major innovation is the application of cutting-edge computational approaches to design point mutations that probe specific questions about collagen assembly and stability. Together, we expect these studies to suggest new avenues for the treatment of collagen associated disorders. Finally, these experiments serve as an exciting training opportunity, providing me with a mentored research experience in inter- disciplinary, translational studies.