DOMAIN MAPPING CLOSTRIDIUM PERFRINGENS THETA TOXIN

描述（改编自申请人摘要）：许多细菌 溶细胞毒素表现出产生它们的两形性 作为可溶性单体，但最终作为膜相关的低聚物。 这些毒素缺乏明显的跨膜结构域，事实上具有相对的跨膜结构域。 亲水结构。 这些蛋白质进行蛋白质合成的机制 从可溶性蛋白质到膜相关复合物的转变 被识别。 不像许多细菌毒素，如白喉毒素或 大肠杆菌素，pH值不作为一个触发器的形成“熔融 小球”或插入中间体。 产气荚膜梭菌溶素O（PFO）是一种溶细胞素（Mr 54，000）由产气荚膜梭菌产生和分泌，属于 相关溶细胞素家族统称为“硫醇活化的 由多种革兰氏阳性病原体产生的”溶细胞素 细菌种类 PFO代表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。 绑定到 在靶膜上，PFO单体寡聚成超分子复合物 然后裂解细胞 PFO如何插入膜和形成孔 成就仍然未知。 本建议旨在确定 位置（即，蛋白质-水溶液、蛋白质-膜或蛋白质-蛋白质） PFO与靶点相互作用前后的不同结构域 膜。 该提案的具体目标是：1）生成 PFOala中的单位点半胱氨酸取代，2） PFO-膜相互作用; 3）鉴定暴露于 在细胞溶解机制的各个阶段的水性介质; 4） 位于界面区域的残基的鉴定 PFO寡聚体的单体亚基;和5）测定PFO寡聚体的 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， 荧光染料如NBD的特异性连接位点，其发射是 对环境的极性很敏感 荧光寿命 与PFO相连的单个染料的强度和碰撞猝灭将 以确定是否有特定的残留物残留在水溶液中 相，移动到脂质双层中，或形成界面的一部分。 在寡聚化PFO中接触的结构域。 由于低聚 仅发生在10 ℃以上，而结合发生在所有温度下，插入 探针标记的残基进入双层可以分配给 结合事件或寡聚化事件。

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.