DIPHTHERIA TOXIN STRUCTURE AND MEMBRANE INTERACTION

白喉毒素结构和膜相互作用

• 批准号: 2176390
• 负责人: Erwin London
• 金额: $ 24.6万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-04-01 至 1998-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2176390
• 关键词: Corynebacterium diphtheriae; binding proteins; biotin; diphtheria toxin; epitope mapping; fluorimetry; immunochemistry; membrane activity; membrane proteins; membrane structure; monoclonal antibody; protein folding; protein structure; protein transport; secretory protein; site directed mutagenesis; structural biology; western blottings

The aim of this project is to understand how proteins fold within and translocate across membranes by studying the behavior of diphtheria toxin, and to apply the lessons learned to other systems. One of our goals in this project will be to determine the structure of the membrane-inserted toxin at medium-to-high resolution, defining not only what parts of the protein are embedded within the bilayer, but also identifying the secondary structure (which may be a combination of beta-sheets and alpha- helices) and orientation of membrane embedded segments. This will involve systematically mapping out of the depth of individual residues in membrane penetration regions of the protein, and identifying whether individual residues in membrane-embedded segments are exposed to lipid, the interior of the protein, or the aqueous solution. These questions are being tackled by a combination of methods, including immunochemical, biotin/streptavidin, fluorescence and especially a fluorescence quenching method to determine depth recently developed in our laboratory. These approaches will be applied to membrane-inserted toxin and to membrane- inserted mutant toxins in which site-directed mutagenesis and chemical labeling are combined to label single residues with fluorescent groups or biotin. The translocation of the toxin across membranes is also being studied. We are using an in vitro translocation system that allows control of translocation conditions to characterize the translocation process and pore formation by the toxin. In additional studies, we will begin to look at the mechanism of membrane translocation of ordinary cellular proteins through studies of the SecA protein. We have already found that SecA, which plays a central role in E. coli translocation, shows significant parallels in its behavior to that of diphtheria toxin. These studies will contribute greatly to our understanding of membrane protein insertion and structure, protein translocation and the behavior of bacterial toxin proteins. The new methods we are developing should be applicable to the analysis of membrane protein structure at medium-to-high resolution in many systems. In addition, these studies should have a significant impact on the design of therapeutically useful "immunotoxin" agents, an area of intense applied biomedical research, and in the development of more effective vaccines and therapeutic agents for bacterial diseases where toxin proteins are important virulence factors.

该项目的目的是了解蛋白质如何在内部折叠 通过研究白喉毒素的行为跨膜易位， 并将学到的经验教训应用到其他系统中。 我们的目标之一是 该项目将确定插入膜的结构 中高分辨率的毒素，不仅定义了毒素的哪些部分 蛋白质嵌入双层内，还可以识别 二级结构（可能是 β-折叠和 α-折叠的组合） 螺旋）和膜嵌入片段的方向。 这将涉及到 系统地绘制出膜中各个残基的深度 蛋白质的渗透区域，并确定个体是否 膜嵌入片段中的残基暴露于脂质，内部 蛋白质或水溶液。 这些问题正在 通过组合方法解决，包括免疫化学、 生物素/链霉亲和素、荧光，尤其是荧光猝灭 我们实验室最近开发的确定深度的方法。 这些 方法将应用于膜插入毒素和膜- 插入突变毒素，其中进行定点诱变和化学 组合标记以用荧光基团标记单个残基或 生物素。 毒素跨膜易位也正在发生 研究过。 我们正在使用体外易位系统，该系统允许 控制易位条件来表征易位 毒素的过程和孔隙形成。 在额外的研究中，我们将 开始研究普通细胞的膜易位机制 通过研究 SecA 蛋白来研究细胞蛋白。 我们已经 发现 SecA 在大肠杆菌易位中发挥核心作用， 其行为与白喉毒素显着相似。 这些研究将极大地促进我们对膜的理解 蛋白质插入和结构、蛋白质易位和行为 细菌毒素蛋白。 我们正在开发的新方法应该是 适用于中高端膜蛋白结构分析 许多系统中的分辨率。 此外，这些研究应该有一个 对治疗有用的“免疫毒素”的设计产生重大影响 药剂，一个密集的应用生物医学研究领域，并且在 开发更有效的疫苗和治疗药物 毒素蛋白是重要毒力因子的细菌性疾病。