Protein Unfolding During Anthrax Toxin Translocation

炭疽毒素易位过程中蛋白质的折叠

• 批准号: 6909009
• 负责人: Bryan Andrew Krantz
• 金额: $ 4.83万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6909009
• 关键词: acid base balance; anthrax; anthrax toxin; bacterial genetics; bioenergetics; bioterrorism /chemical warfare; chemical kinetics; conformation; enzyme activity; enzyme mechanism; fluorescence resonance energy transfer; gene mutation; postdoctoral investigator; protein binding; protein folding; protein structure function; protein transport; site directed mutagenesis; stop flow technique; thermodynamics

DESCRIPTION (provided by applicant): Bacterial pathogens have evolved a general strategy of infecting host organisms by means of toxin protein complexes that pattern the AIB mechanism. The active, enzymatic, A moiety binds the receptor, B moiety; the receptor moiety then acts as a vehicle that delivers the A moiety to the cytoplasm from an isolated compartment separated by a membrane bilayer. The enzymatic effector, by means of its specific catalytic activity, then disables the normal physiology of the cell. The tripartite toxin of Bacillus anthracis relies on a "molecular syringe" B moiety, Protective Antigen (PA), which forms a heptad ring structure with a narrow central cavity at neutral extracellular pH. Two different A moiety effectors, Lethal Factor (LF) and Edema Factor (EF), bind PA heptamer. Immediately following endocytosis of membrane bound PA/LF/EF complexes, endosomes acidify, triggering the synchronized formation of a membrane piercing pore form of PA heptamer that presumably injects LF and EF into the cytosol. The narrow central cavity of PA heptamer is too small to accommodate fully native LF or EF, invoking the possibility that translocation is mitigated by protein unfolding. Indeed, stabilized fusions of LF validate this type of mechanism. Modern advances in a variety of fluorescence spectroscopy and imaging methods have enabled the detailed elucidation of protein folding pathways even at the single molecule level. A thorough structural, energetic, and kinetic understanding of the sequence of events leading up to and immediately following translocation using fluorescence methods will enable improved discourse toward the design and implementation of potential therapies for intoxication, including designed protein inhibitors and drugs.

描述（由申请人提供）： 细菌病原体已经进化出一种通过毒素蛋白复合物感染宿主生物体的一般策略，该复合物模拟了 AIB 机制。活性酶 A 部分与受体 B 部分结合；然后受体部分充当载体，将 A 部分从由双层膜分隔的隔离室递送至细胞质。酶效应子通过其特定的催化活性，使细胞的正常生理功能失效。炭疽杆菌的三联毒素依赖于“分子注射器”B部分，即保护性抗原（PA），它在中性细胞外pH下形成具有狭窄中心空腔的七联环结构。两种不同的 A 部分效应子，致死因子 (LF) 和水肿因子 (EF)，结合 PA 七聚体。膜结合的 PA/LF/EF 复合物被内吞后，内体立即酸化，触发 PA 七聚体的膜穿刺孔形式的同步形成，这可能将 LF 和 EF 注入细胞质中。 PA 七聚体狭窄的中央腔太小，无法容纳完全天然的 LF 或 EF，因此可能会因蛋白质解折叠而减轻易位。事实上，LF 的稳定融合验证了这种机制。现代各种荧光光谱和成像方法的进步使得即使在单分子水平上也能够详细阐明蛋白质折叠途径。使用荧光方法对易位之前和之后的事件序列进行彻底的结构、能量和动力学理解，将有助于改进对潜在中毒疗法的设计和实施的讨论，包括设计的蛋白质抑制剂和药物。