STRUCTURAL CHARACTERIZATION OF THROMBOSPONDIN-1 CONSTRUCTS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A polymorphism in Thrombospondin-1 (TSP-1) results in a substitution of a serine for a conserved asparagine at residue 700 in the calcium-binding region, and has been associated with premature familial coronary artery disease. The crystal structure of a related protein, TSP-2, that included this conserved calcium-binding region was solved in our lab. The solution revealed that the asparagine was involved in coordinating a calcium ion, and the presence of a serine was hypothesized to disrupt this interaction. Fluorescence spectroscopy of baculovirus-generated TSP-1 constructs that contain the entire calcium binding regions or various truncations shows that the polymorphism does alter the conformation of the calcium-binding region. In addition, polymorphic proteins bind less calcium with significantly slower kinetics. These data raise several questions, such as how does the presence of the polymorphism alter the structure of the calcium-binding region, what is the structure of the calcium-binding region in the absence of calcium, and how the does structure of the calcium-binding region transition from unbound to the calcium-bound state. In collaboration with the NMR Facility on campus, we propose to answer these questions with NMR spectroscopy, in order to further understand the structural consequences of a TSP-1 polymorphism associated with coronary artery disease.