Mechanism of Protein Localization in Escherichia coli

描述（由申请人提供）：我们的总体目标是使用大肠杆菌作为一个简单的遗传和生化系统来阐明蛋白质跨生物膜易位的分子细节。我们将专注于一个中心组件，SecA ATP酶，它与所有关键组件相互作用，其易位ATP酶活性和膜循环行为的能量学和易位机制的基础。提出了三个具体目标。(1)为了理解SecA信号肽识别的分子基础，SecA信号肽结合位点将通过荧光共振能量转移方法进行映射，并进行竞争和突变研究以确认结合位点分配并阐明该系统中负责简并但特异性肽结合的分子模式。(2)为了理解SecA-SecYEG相互作用和SecA膜循环的结构和功能基础，将分离和表征在这些过程中有缺陷的secA突变体，并且将采用体内SecA膜拓扑学研究来精确定位SecA ATP结合位点周围的SecYEG通道可接近区域和控制SecA膜循环的氨基末端延伸臂。(3)为了阐明SecA ATP酶反应循环，将进行“空闲”和主动易位条件下SecA-SecYEG蛋白脂质体复合物的预稳态和稳态分析。平行研究也将进行功能，突变SecA蛋白与显着降低易位ATP酶活性。这些研究应该提供新的见解逐步操作的SecA nanoparticle和化学机械耦合前蛋白识别，SecYEG通道激活和进行性蛋白质易位。他们应该导致一个详细的了解Sec-dependent蛋白质易位在分子水平上，他们应该有广泛的意义，以了解这些过程中的并行系统，工程这样的途径，并开发新的抗菌剂。

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.