STRUCTURAL CHARACTERIZATION OF THROMBOSPONDIN-1

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 凝血酶敏感蛋白-1(TSP-1)的多态性导致钙结合区第700位的丝氨酸取代保守的天冬酰胺，并与早产家族性冠状动脉疾病有关。在我们的实验室中，一个相关的蛋白质TSP-2的晶体结构被破解，它包括这个保守的钙结合区域。该解决方案显示，天冬酰胺参与协调一个钙离子，并假设丝氨酸的存在，以破坏这种相互作用。对杆状病毒产生的包含完整钙结合区或各种截断的TSP-1结构的荧光光谱表明，该多态性确实改变了钙结合区的构象。此外，多态蛋白质结合的钙较少，动力学明显较慢。这些数据提出了几个问题，如多态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非结合状态转变为钙结合状态。在与校园核磁共振设施的合作中，我们建议用核磁共振波谱来回答这些问题，以进一步了解TSP-1基因多态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.