Mechanism of Protein Localization in Escherichia coli

描述(由申请人提供)：我们的总体目标是利用大肠杆菌作为一种简便的遗传和生化系统来阐明蛋白质跨生物膜转运的分子细节。我们将重点关注与所有关键成分相互作用的核心成分--SecA-ATPase，它的转位ATPase活性和膜循环行为是能量学和转位机制的基础。提出了三个具体目标。(1)为了了解SecA信号肽识别的分子基础，将用荧光共振能量转移方法绘制SecA信号肽的结合位点图，并进行竞争和突变研究，以确认结合位点的分配，并阐明该系统中简并但特异的肽结合的分子模式。(2)为了了解SecA-SecYEG相互作用和SecA膜循环的结构和功能基础，我们将分离和鉴定在这些过程中存在缺陷的SecA突变体，并利用体内的SecA膜拓扑结构研究来精确定位控制SecA膜循环的SecA ATP结合位点和氨基末端延伸臂周围的SecYEG通道可及区域。(3)为了阐明SecA ATPase的反应循环，将对SecA-SecYEG蛋白脂质体复合体在“空转”和主动转运条件下的稳态和稳态进行分析。同时，还将对易位ATPase活性显著降低的功能性突变的SecA蛋白进行平行研究。这些研究将为SecA纳米运动的逐步操作及其与前蛋白识别、SecYEG通道激活和进行性蛋白质易位的化学机械耦合提供新的见解。它们将导致在分子水平上对SEC依赖的蛋白质易位的详细理解，并且对于理解平行系统中的这些过程、设计这样的通路以及开发新型抗菌药物具有广泛的意义。

DESCRIPTION (provided by applicant): Our overall goal is to elucidate the molecular details of protein translocation across biological membranes using Escherichia coli as a facile genetic and biochemical system. We will focus on a central component, SecA ATPase, which interacts with all key components, and whose translocation ATPase activity and membrane cycling behavior underlie the energetics and translocation mechanism. Three specific aims are proposed. (1) To understand the molecular basis for SecA-signal peptide recognition, the SecA signal peptide-binding site will be mapped by fluorescence resonance energy transfer methodology, and competition and mutational studies will be undertaken to confirm the binding site assignment and elucidate the molecular modes responsible for degenerate but specific peptide binding in this system. (2) To understand the structural and functional bases of SecA-SecYEG interaction and SecA membrane cycling, secA mutants defective in these processes will be isolated and characterized, and in vivo SecA membrane topology studies will be employed to pinpoint SecYEG channel-accessible regions around the SecA ATP- binding site and amino-terminal extension arm that control SecA membrane cycling. (3) To elucidate the SecA ATPase reaction cycle, pre-steady-state and steady-state analyses of a SecA-SecYEG proteoliposome complex under "idling" and actively-translocating conditions will be undertaken. Parallel studies will also be undertaken with functional, mutant SecA proteins with remarkably reduced translocation ATPase activity. These studies should provide novel insights into the stepwise operation of the SecA nanomotor and its chemo-mechanical coupling to preprotein recognition, SecYEG channel activation and processive protein translocation. They should lead to a detailed understanding of Sec-dependent protein translocation at the molecular level, and they should be of broad significance to understand these processes in parallel systems, to engineer such pathways, and to develop novel anti-bacterial agents.