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

项目摘要 蛋白质跨膜转运复杂过程的机制 是出了名的难以定义目前，很少有模型系统存在，其中该过程已经 这一问题没有得到充分的描述，近年来在这一问题上严重缺乏进展。之一 导致这一领域缺乏发展的一个因素是缺乏结构数据， 蛋白质转运过程中的中间产物。这在很大程度上是由于在获得纯， 在这些国家的同质样本。随着高分辨率低温电子显微镜（cryo-EM）的出现， 以及旨在解决这些问题的复杂数据处理算法的开发，我们 现在能够在解决这一问题方面取得重大进展。在这个奖项下，我将描述 利用一种被称为梭菌的分泌性细菌毒素进行蛋白质移位的基本过程 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 结构和生物化学训练的基础以及我收集的初步数据 主题，包括五个不同的结构的CDT，我已经解决了冷冻电镜。这些结构将 用于指导开发新的工具和分析，以严格描述CDT易位机制。 我将解决的悬而未决的问题，如何孔形成的调节，并确定什么因素驱动 在这个模型系统中的蛋白质易位导致几个关键易位的结构的阐明 在这个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， 在中毒的情况下定义蛋白质易位机制。我提出的基于细胞的检测将 形成了一个重要组成部分，我的一个额外的一年博士后培训根据这个奖项的指导博士。 在此期间，我将获得维护和处理哺乳动物组织的专业知识 培养细胞系用于各种下游应用。我将利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仪，利用承诺强大的研究环境， 到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 这是我打算在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 将继续探索使用细菌分泌系统的蛋白质易位机制的现象 作为一种工具我相信我丰富的结构生物学和生物化学背景， 作为研究生导师的经历使我非常适合这样的角色。

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.