STRUCTURAL CHARACTERIZATION OF THE ANTHRAX TOXIN PROTECTIVE ANTIGEN

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。子项目及 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。 由于炭疽被用作生物恐怖主义制剂的持续威胁，有必要开发在感染的任何阶段阻断炭疽毒素作用的疗法。本研究的目的是了解pH如何控制保护性抗原（PA）中的大构象变化以形成跨膜孔，这是启动与炭疽相关的细胞毒性的必要步骤，这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。对这一假设的支持来自初步实验的结果，该结果显示2-氟组氨酸（2-FHis）（其具有显著较低的侧链pKa（pKa ~1））均匀生物合成掺入PA的七聚体中导致不能经历导致孔的pH依赖性变化的多聚体蛋白质结构。我们的具体目标是： 1.确定受体调节孔形成的结构基础。基于初步结果，我们假设，组氨酸残基的质子化，特别是在PA受体结合结构域（域4）引起的构象变化，导致损失的结合受体。我们计划使用生物物理方法，包括荧光，圆二色谱和NMR分离和表征PA的受体结合结构域的结构。 2.鉴定PA中发生pH依赖性结构变化导致孔形成的特定区域。除了在PA结构域2中发生的已知结构变化之外，结构变化可能在整个蛋白质中发生，这是促进功能孔正确形成所需的。将使用溶液（[13 C]和[19 F]）和固态NMR确定这些结构变化如何取决于pH。

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.