STRUCTURAL CHARACTERIZATION OF THROMBOSPONDIN-1 CONSTRUCTS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 血小板反应蛋白-1（TSP-1）的多态性导致钙结合区第700位残基上的保守天冬酰胺被丝氨酸取代，并与家族性早发性冠状动脉疾病相关。 在我们的实验室中解决了一个相关蛋白质TSP-2的晶体结构，其中包括这个保守的钙结合区域。 该溶液揭示了天冬酰胺参与了钙离子的配位，并且假设丝氨酸的存在破坏了这种相互作用。 荧光光谱的杆状病毒产生的TSP-1的结构，包含整个钙结合区或各种截断显示，多态性改变的钙结合区的构象。 此外，多态性蛋白质结合较少的钙，动力学明显较慢。 这些数据提出了几个问题，如多态性的存在如何改变钙结合区的结构，在没有钙的情况下钙结合区的结构是什么，以及钙结合区的结构如何从未结合状态过渡到钙结合状态。 与校园内的NMR设施合作，我们建议用NMR光谱来回答这些问题，以进一步了解TSP-1多态性与冠状动脉疾病相关的结构后果。

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.