These studies will provide insight into molecular interactions and conformational changes that are involved in conversion of a soluble protein, fibronectin, to insoluble fibrils of the extracellular matrix. The conversion as well as its product is, despite years of research effort, still poorly understood. This is mainly due to the lack of control over the conversion, and the difficulties that are associated with investigations of the resulting heterogeneous aggregates. The project will take advantage of an experimentally more tractable model system that employs a small fragment of fibronectin, termed anastellin. Anastellin binds to soluble fibronectin and converts it to an insoluble form that morphologically and functionally resembles fibronectin fibrils deposited by cells. The binding sites for anastellin in fibronectin include the first through third and the eleventh type III (FN3) domains. To investigate the structure and dynamics of the complexes between anastellin and its target FN3 domains, hydrogen/deuterium exchange, relaxation dispersion experiments, proteolysis and truncations will be used in conjunction with nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography. These studies will elucidate the molecular basis of the interactions that play key roles in this model assembly process, and characterize how unfolding of the interacting FN3 domains contributes to fibril formation. Based on the observed structural and dynamic changes in FN3 domains that are induced by anastellin binding, a model will be formulated for assembly of fibronectin aggregates. In addition, a novel method that will hinder aggregation of proteins during solution state NMR experiments will be developed and tested.
Broader Impacts This research will help to elucidate how molecular interactions and conformational changes lead to conversion of soluble fibronectin to insoluble fibrils. In addition, a new method will be developed for investigations of aggregation-prone proteins by solution state NMR experiments. These studies will provide unique opportunities for graduate, undergraduate and high school students to get involved in biochemical and structural biology research. Special emphasis will be placed on recruitment of Native American students. The principal investigator will also develop a seminar for freshmen that will discuss exciting biological discoveries that center on proteins. This seminar will aim to inspire and motivate first year students in order to increase their retention. In addition it will provide an excellent opportunity to get students interested in biochemical research early on during their studies. Finally, the principal investigator will introduce biochemical applications into an undergraduate physical chemistry course for non-majors in order to expose a broader audience of students to this interesting subject.
