ANALYZE THE COMPLEX PROTEIN ASSEMBLY USING SAXS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 我们的实验室专注于蛋白质-蛋白质相互作用的分子基础，这对人类正常和病理反应中的细胞信号转导至关重要。目前，我们在两个主要领域开展工作。首先是人蛋白酶、胰岛素降解酶（IDE）与其底物的相互作用。IDE是一种110 kDa的金属蛋白酶，参与胰岛素和淀粉样蛋白的清除，这些肽对糖尿病和阿尔茨海默病的进展至关重要。我们已经使用SAXS探测底物结合调节的开-闭构象转换以及IDE寡聚化的变构调节。我们最近发现巨噬细胞炎性蛋白（MIP）-1 <$和MIP-1 <$作为IDE的新底物。MIP-1 <$和MIP-1 <$形成高分子量聚集体。我们使用SAXS证明MIP-1 <$和MIP-1 <$在溶液中形成棒状多分散聚合物。我建议继续使用SAXS来了解IDE及其底物的结构特征以及它们如何相互作用以实现其生物功能。第二个是引起炭疽的细菌分泌的毒素的相互作用。炭疽菌还分泌>200种分泌因子。这包括三种主要的炭疽毒素，保护性抗原（PA），水肿因子（EF）和致死因子（LF）以及PA-EF和PA-LF的蛋白复合物。我们使用SAXS分析作为一种方法来探测纯化的炭疽毒素和毒素复合物的结构特征。我们还选择了10个这样的因素，包括孔形成毒素，蛋白酶和细胞壁修饰酶，并将应用SAXS探测这些毒力因子的生物物理特性。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Our lab focuses on the molecular basis of protein-protein interaction crucial for cell signal transduction in the normal and pathologic responses of humans. Currently, we work on two major areas. First is the interaction of human protease, insulin-degrading enzyme (IDE) with its substrates. IDE, a 110 kDa metalloprotease, is involved in the clearance of insulin and amyloid ¿, peptides crucial for the progression of diabetes and Alzheimers' disease. We have used SAXS to probe the open-closed conformational switch regulated by substrate binding as well as the allosteric regulation by the oligomerization of IDE. We recently identify macrophage inflammatory protein (MIP)-1¿ and MIP-1¿ as the novel substrate of IDE. MIP-1¿ and MIP-1¿ form high molecular weight aggregates. We used SAXS to demonstrate that MIP-1¿ and MIP-1¿ form the rod-shaped polydisperse polymers in solution. I propose to continue to use SAXS to understand the structure features of IDE and its substrates and how they interact to achieve their biological functions. Second is the interactions of toxins secreted by bacteria that cause anthrax. Anthrax bacteria also secrete >200 secreted factors. This includes three major anthrax toxins, protective antigen (PA), edema factor (EF), and lethal factor (LF) and the protein complex of PA-EF and PA-LF. We use SAXS analysis as a method to probe the structural features of purified anthrax toxins and toxin complex. We have also chosen 10 of such factors including pore-forming toxins, proteases, and cell wall modifying enzymes and will apply SAXS to probe the biophysical properties of these virulence factors.