STRUCTURAL CHARACTERIZATION OF THROMBOSPONDIN-1 CONSTRUCTS

血小板反应蛋白-1 构建体的结构表征

• 批准号: 7721624
• 负责人: DEANE Fremont MOSHER
• 金额: $ 0.6万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-03-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7721624
• 关键词: Asparagine; Baculoviruses; Calcium; Calcium Binding; Calcium ion; Collaborations; Computer Retrieval of Information on Scientific Projects Database; Coronary Arteriosclerosis; Data; Fluorescence Spectroscopy; Funding; Genetic Polymorphism; Grant; Institution; Kinetics; Molecular Conformation; NMR Spectroscopy; Protein Binding; Proteins; Research; Research Personnel; Resources; Serine; Solutions; Source; Structure; Thrombospondin 1; United States National Institutes of Health; thrombospondin 2

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 Thrombospondin-1 (TSP-1) 的多态性导致钙结合区第 700 位残基上的丝氨酸取代保守的天冬酰胺，并且与早产家族性冠状动脉疾病有关。 我们的实验室解析了包含此保守钙结合区域的相关蛋白质 TSP-2 的晶体结构。 该溶液显示天冬酰胺参与钙离子的配位，并且推测丝氨酸的存在会破坏这种相互作用。 杆状病毒生成的含有整个钙结合区或各种截断的 TSP-1 构建体的荧光光谱表明，多态性确实改变了钙结合区的构象。 此外，多态蛋白结合的钙较少，动力学明显较慢。 这些数据提出了几个问题，例如多态性的存在如何改变钙结合区域的结构，在没有钙的情况下钙结合区域的结构是什么，以及钙结合区域的结构如何？结合区域从未结合状态转变为钙结合状态。 我们与校园内的 NMR 设施合作，建议用 NMR 波谱来回答这些问题，以便进一步了解与冠状动脉疾病相关的 TSP-1 多态性的结构后果。