STRUCTURAL CHARACTERIZATION OF THE ANTHRAX TOXIN PROTECTIVE ANTIGEN

炭疽毒素保护性抗原的结构表征

• 批准号: 8359660
• 负责人: James G. Bann
• 金额: $ 26.11万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8359660
• 关键词: Anthrax disease; Antigen Receptors; Antigens; Binding; Bioterrorism; Circular Dichroism Spectroscopy; Development; Fluorescence; Funding; Grant; Histidine; Infection; Membrane; Methods; National Center for Research Resources; Principal Investigator; Proteins; Research; Research Infrastructure; Resources; Side; Solutions; Source; Staging; Structure; Therapeutic; United States National Institutes of Health; anthrax toxin; base; cost; cytotoxicity; protein structure; protein structure function; protonation; receptor; receptor binding; research study; solid state nuclear magnetic resonance; therapy development

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. 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.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 使用炭疽作为生物恐怖剂的持续威胁需要开发疗法，以阻止在任何感染阶段的炭疽毒素作用。这项研究的目的是了解pH如何控制保护性抗原（PA）形成膜毛孔的巨大构象变化，这是启动与炭疽相关的细胞毒性的必要步骤，这将指导针对抗炭疽感染的有效治疗剂的发展。具体假设是，在受体存在下形成孔的形成取决于一个或多个特定的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，这些实验表明，2-氟化鼠（2-FHIS）的均匀生物合成掺入（其在多上蛋白质结构中，PA的heptamer均无法使pH依赖性变化，这使PA的hepTamer均在PA的hepTAMER中具有较低的侧链PKA（PKA〜1）的显着下降。我们的具体目的是： 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在PA受体结合结构域（结构域4）的质子化会导致构象变化，从而导致与受体结合的丧失。我们计划使用生物物理方法（包括荧光，圆形二色性光谱和NMR）分离和表征PA受体结合结构域的结构。 2。确定PA内经历pH依赖性结构变化的特定区域，从而导致孔形成。除了PA的域2中发生的已知结构变化外，在整个蛋白质中可能会发生结构变化，以促进功能孔的正确形成。这些结构变化如何取决于pH，使用溶液（[13C]和[19F]）和固态NMR确定。