STRUCTURAL CHARACTERIZATION OF THE ANTHRAX TOXIN PROTECTIVE ANTIGEN

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。 使用炭疽作为生物恐怖主义制剂的持续威胁要求开发在感染的任何阶段阻断炭疽毒素作用的治疗方法。本研究的目的是了解pH如何调控保护性抗原(PA)的大量构象变化以形成跨孔的膜，这是启动炭疽相关细胞毒性的必要步骤，将指导针对炭疽感染的有效治疗药物的开发。具体的假设是，在受体存在的情况下，孔的形成严重依赖于一个或多个特定组氨酸残基的质子化。支持这一假说的初步实验结果表明，2-氟组氨酸(2-FHIS)(侧链明显较低的PKA(PKA~1))均匀地生物合成掺入PA的七聚体中，导致了一种多聚体的蛋白质结构，而这种多聚体结构不能经历导致气孔的pH依赖的变化。我们的具体目标是： 1.确定受体调节孔道形成的结构基础。根据初步结果，我们假设PA受体结合域(结构域4)中组氨酸残基的质子化引起构象变化，导致与受体结合的丧失。我们计划利用生物物理方法，包括荧光、圆二色谱和核磁共振，分离和表征PA的受体结合域的结构。 2.确定PA内经历与pH有关的结构变化导致孔隙形成的特定区域。除了已知的在PA的结构域2中发生的结构变化外，整个蛋白质可能也会发生结构变化，这是促进功能孔正确形成所必需的。这些结构变化如何依赖于pH将使用溶液([13C]和[19F])和固态核磁共振来确定。

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. The ongoing threat of the use of anthrax as a bioterrorism agent necessitates the development of therapies that block the action of anthrax toxin at any stage of infection. The objective of this research is to understand how pH governs the large conformational change in the protective antigen (PA) to form a membrane spanning pore, a requisite step to initiating the cytotoxicity associated with anthrax, which will guide the development of effective therapeutics directed against anthrax infection. The specific hypothesis is that formation of a pore in the presence of the receptor is critically dependent on the protonation of one or more specific histidine residues. Support for this hypothesis derives from results of preliminary experiments which show that the uniform biosynthetic incorporation of 2-fluorohistidine (2-FHis) (which has a dramatically lower side-chain pKa (pKa ~1)) into the heptamer of PA results in a multimeric protein structure that cannot undergo the pH dependent changes leading to a pore. Our specific aims are to: 1. Determine the structural basis by which the receptor modulates pore formation. Based on preliminary results, we hypothesize that protonation of histidine residues specifically in the PA receptor binding domain (domain 4) causes a conformational change that results in a loss of binding to the receptor. We plan to isolate and characterize the structure of the receptor binding domain of PA using biophysical methods, including fluorescence, circular dichroism spectroscopy and NMR. 2. Identify specific regions within PA that undergo pH-dependent structural changes that result in pore formation. In addition to the known structural changes that occur in domain 2 of PA, structural changes are likely to occur throughout the protein that are required to facilitate the correct formation of a functional pore. How these structural changes are dependent upon pH will be determined using solution ([13C] and [19F]) and solid-state NMR.