DOMAIN MAPPING CLOSTRIDIUM PERFRINGENS THETA TOXIN

描述(改编自申请者的摘要)：许多细菌 细胞溶解毒素在其产生时表现出一种双晶型的性质 作为可溶单体，但最终以膜相关低聚物的形式结束。 这些毒素缺乏明显的跨膜结构域，实际上具有相对 亲水性结构。这些蛋白质执行该功能的机制 从可溶性蛋白质到膜结合复合体的转变并没有 已被确认身份。与许多细菌毒素不同，如白喉毒素或 粘菌素，pH值并不是形成熔化的触发因素 球状“或插入中间体。红细胞溶血素O(PFO)，一种细胞溶血素(MR 54,000)由产气荚膜梭菌产生和分泌，属于 相关的细胞溶血素家族统称为“硫醇激活的 细胞溶血素是由多种革兰氏阳性致病菌产生的 细菌种类。PFO是一种具有亲水性的细胞溶解毒素 初级结构，但形成溶细胞膜复合体。绑定到后 目标膜、PFO单体齐聚成超分子络合物 然后裂解细胞。PFO的膜插入和成孔是怎样的 是否成功仍是未知数。该提案旨在确定 位置(即蛋白质-水、蛋白质-膜或蛋白质-蛋白质) PFO与靶相互作用前后的不同结构域 膜。这项提议的具体目标是：1)产生 PFOala中的单位半胱氨酸取代，2)鉴定 PFO-膜相互作用；3)PFO暴露区域的鉴定 细胞溶解机制不同阶段的水介质；4) 分子间界面域上残基的鉴定 PFO齐聚物的单体亚基；以及5)测定 在细胞溶解过程的不同阶段，PFO插入细胞膜。 独特的半胱氨酸将被放置在PFO的一级结构中，作为 荧光染料的特定附着位置，如NBD，其发射是 对环境的极性很敏感。荧光寿命 PFO Will上单一染料的强度和碰撞猝灭 被监测以确定特定残留物是否为水溶液 相，移动到脂质双层，或形成界面的一部分 齐聚PFO中接触的结构域。自齐聚以来 只发生在10℃以上，而结合在所有温度下都发生，插入 标记残基进入双层的探针可以被分配给 结合事件或寡聚事件。

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.