DOMAIN MAPPING CLOSTRIDIUM PERFRINGENS THETA TOXIN

产气荚膜梭菌 Theta 毒素的结构域图谱

• 批准号: 2672473
• 负责人: Rodney K. Tweten
• 金额: $ 19.32万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-04-01 至 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2672473
• 关键词: Clostridium; chemical binding; crosslink; cysteine; cytolysins; cytotoxicity; fluorescence spectrometry; fluorescent dye /probe; intermolecular interaction; mutant; posttranslational modifications; protein sequence; site directed mutagenesis; tetanus toxin; toxin metabolism

DESCRIPTION (Adapted from the applicant's abstract): Many bacterial cytolytic toxins exhibit an amphimorphic nature in which they are produced as soluble monomers, but ultimately end up as membrane-associated oligomers. These toxins lack obvious transmembrane domains and in fact have relatively hydrophilic structures. The mechanism by which these proteins carry out the transition from a soluble protein to a membrane associated complex has not been identified. Unlike many bacterial toxins, such as diphtheria toxin or the colicins, pH does not act as a trigger for the formation of a "molten globule" or insertion intermediate. Perfringolysin O (PFO), a cytolysin (Mr 54,000) produced and secreted by Clostridium perfringens, belongs to a family of related cytolysins collectively called the "thiol-activated cytolysins" that are produced by a variety of gram positive pathogenic bacterial species. PFO typifies a cytolytic toxin which has a hydrophilic primary structure but forms a cytolytic membrane complex. After binding to the target membrane, PFO monomers oligomerize into supramolecular complexes and lyse the cell. How membrane insertion and pore formation by PFO is accomplished remains unknown. This proposal is designed to identify the location (i.e., protein-aqueous, protein-membrane, or protein-protein) of various domains of PFO before and after its interaction with target membranes. The specific aims of this proposal are: 1) generation of single-site cysteine substitutions in PFOala, 2) identification of PFO-membrane interactions; 3) identification of regions of PFO exposed to the aqueous medium at various stages of the cytolytic mechanism; 4) identification of residues located at the interfacial domains of the monomeric subunit of the PFO oligomer; and 5) determination of the extent of PFO insertion into the membrane at various stages of the cytolytic process. Unique cysteines will be placed into the primary structure of PFO to act as specific attachment sites for fluorescent dyes such as NBD whose emission is sensitive to the polarity of their environment. The fluorescence lifetime intensity and collisional quenching of the single dye attached to PFO will be monitored to determine if a specific residue remains is the aqueous phase, moves into the lipid bilayer, or forms part of the interfacial domains that are in contact in the oligomerized PFO. Since oligomerization only occurs above 10C, whereas binding occurs at all temperatures, insertion of a probe labeled residue into the bilayer can be assigned to either the binding event or the oligomerization event.

描述（改编自申请人的摘要）：许多细菌 溶细胞毒素在其产生过程中表现出两性性质 作为可溶性单体，但最终成为膜相关的低聚物。 这些毒素缺乏明显的跨膜结构域，实际上具有相对 亲水结构。 这些蛋白质执行的机制 从可溶性蛋白质到膜相关复合物的转变尚未发生 已被识别。 与许多细菌毒素不同，例如白喉毒素或 大肠杆菌素、pH 值不会引发“熔解”的形成。 球”或插入中间体。Perfringolysin O (PFO)，一种溶细胞素（Mr 54,000）由产气荚膜梭菌产生和分泌，属于 相关的溶细胞素家族统称为“硫醇激活的 多种革兰氏阳性致病菌产生的溶细胞素 细菌种类。 PFO 是一种具有亲水性的溶细胞毒素。 一级结构，但形成溶细胞膜复合物。 绑定后 目标膜上，PFO 单体寡聚成超分子复合物 并裂解细胞。 PFO 的膜插入和孔形成是如何进行的 完成情况仍未知。 该提案旨在确定 的位置（即蛋白质-水、蛋白质-膜或蛋白质-蛋白质） PFO 与目标相互作用前后的各个域 膜。 该提案的具体目标是：1） PFOala 中的单位点半胱氨酸取代，2) 鉴定 PFO-膜相互作用； 3) 识别 PFO 暴露区域 溶细胞机制各个阶段的水介质； 4） 位于界面域的残基的鉴定 PFO寡聚体的单体亚基； 5) 确定程度 PFO 在细胞溶解过程的各个阶段插入膜中。 独特的半胱氨酸将被置于 PFO 的一级结构中，以充当 荧光染料（例如 NBD）的特定附着位点，其发射量为 对环境的极性敏感。 荧光寿命 附着在 PFO 上的单一染料的强度和碰撞猝灭将 进行监测以确定特定残留物是否残留在水相中 相，进入脂质双层，或形成界面的一部分 寡聚 PFO 中接触的结构域。 由于低聚 仅发生在 10°C 以上，而结合发生在所有温度下，插入 探针标记残基进入双层可以分配给 结合事件或寡聚化事件。