Defining the mechanism of protein translocation using a bacterial exotoxin as a model system

项目总结 蛋白质跨质膜转运复杂过程的机制 一直是出了名的难以定义。目前，很少有模型系统在其中进行过这一过程 对这一问题进行了充分的描述，近年来在这一问题上严重缺乏进展。其中一个 导致这一领域缺乏发展的因素是缺乏描述关键问题的结构数据 发生在蛋白质转位过程中的中间产物。这在很大程度上是由于获得纯净的， 同质的，这些状态下的样本。随着高分辨率低温电子显微镜(CRYO-EM)的出现 以及为解决这些问题而设计的复杂数据处理算法的开发，我们 我们现在能够在解决这一问题上取得重大进展。在这个奖项下，我将描述 利用一种被称为梭状芽胞杆菌的分泌型细菌毒素进行蛋白质转运的基本过程 艰难梭菌转移酶毒素(CDT)作为模型系统。我在这里提出的实验将建立在我强大的 结构和生化训练的基础以及我收集的有关这方面的初步数据 主题，包括我已经通过冷冻-EM解决的CDT的五个不同结构。这些结构将是 用于指导开发新的工具和检测方法，以严格描述CDT转位机制。 我将解决如何调节孔隙形成的悬而未决的问题，并确定驱动因素是什么。 这个模型系统中的蛋白质易位导致了几个关键易位结构的阐明 在这一过程中存在的中间体。这些结构将在体外和组织培养中进行探测 在醉酒的情况下定义蛋白质转运机制。我提议的基于细胞的分析将 作为我额外一年的博士后培训的重要组成部分，我在这个奖项的指导下，接受了博士后培训。 在此期间，我将获得维护和处理哺乳动物组织的专业知识 培养各种下游应用的细胞系。我将使用这些基础知识来建立我的专业知识 共聚焦显微镜和流式细胞术，利用承诺的强大研究环境 在范德比尔特大学医学中心接受培训。我将在这个奖项下接受的培训将增加一个新的 我打算在一家R1机构开发的独立研究项目的一个方面。我实验室的重点是 将继续利用细菌分泌系统探索蛋白质转位机制的现象 作为一种工具。我相信我博大精深的结构生物学和生物化学背景，加上我广博的 作为研究生导师的经历使我非常适合担任这样的角色。

PROJECT SUMMARY The mechanisms underlying the complex process of protein translocation across a plasma membrane have been notoriously difficult to define. Currently, few model systems exist in which this process has been adequately described, with a serious lack of progress being made on this subject in recent years. One of the factors that has led to a lack of development in this area is the absence of structural data which describe key intermediates that occur during protein translocation. This is largely due to complications in obtaining pure, homogenous, samples in these states. With the advent of high-resolution cryo electron microscopy (cryo-EM) and the development of sophisticated data processing algorithms that are designed to tackle these issues, we are now in a position to make significant progress in addressing this question. Under this award, I will describe the fundamental process of protein translocation using a secreted bacterial toxin known as the Clostridioides difficile transferase toxin (CDT) as a model system. The experiments I propose here will build upon my strong foundation of structural and biochemical training as well as the preliminary data that I have collected on this subject, including five distinct structures of CDT that I have already solved by cryo-EM. These structures will be used to guide the development of novel tools and assays to rigorously describe CDT translocation mechanisms. I will address the outstanding questions of how pore formation is regulated and determine what factors drive protein translocation in this model system leading to the elucidation of several structures of key translocation intermediates that exist during this process. These structures will be probed both in vitro and in tissue culture to define protein translocation mechanisms in the context of intoxication. The cell based assays I am proposing will form a crucial component of my one additional year of postdoctoral training under this award as guided by Dr. Borden Lacy during which time I will gain expertise in the maintenance and processing of mammalian tissue culture cell lines for a variety of downstream applications. I will use this base knowledge to build my expertise in confocal microscopy and flow cytometry, taking advantage of the strong research environment at commitment to training at Vanderbilt University Medical Center. The training that I will receive under this award will add a new facet to the independent research program that I intend to develop at a R1 institution. The focus of my lab will be to continue probing the phenomenon of protein translocation mechanisms using bacterial secretion systems as a tool. I believe my extensive structural biology and biochemical background coupled with my extensive experience as a mentor to graduate students make me well suited for such a role.