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个简单的遗传和生化系统阐明蛋白质跨生物膜易位的分子细节。我们将重点关注一个中心成分，SecA atp酶，它与所有关键成分相互作用，其易位atp酶活性和膜循环行为是能量学和易位机制的基础。提出了三个具体目标。(1)为了解SecA信号肽识别的分子基础，将采用荧光共振能量转移方法绘制SecA信号肽结合位点，并进行竞争和突变研究，以确定结合位点的分配，阐明该系统中退化但特异性肽结合的分子模式。(2)为了了解SecA-SecYEG相互作用和SecA膜循环的结构和功能基础，将分离和表征在这些过程中存在缺陷的SecA突变体，并利用体内SecA膜拓扑研究来确定控制SecA膜循环的SecA ATP结合位点和氨基末端延伸臂周围的SecYEG通道可达区域。(3)为了阐明SecA atp酶的反应周期，将进行SecA- secyeg蛋白脂质体复合物在“空转”和主动易位条件下的预稳态和稳态分析。同时，还将对具有显著降低易位atp酶活性的功能性突变SecA蛋白进行平行研究。这些研究将为SecA纳米马达的逐步运作及其化学-机械耦合对前蛋白识别、SecYEG通道激活和蛋白易位的影响提供新的见解。它们应该导致在分子水平上对sec依赖性蛋白质易位的详细理解，并且它们应该具有广泛的意义，以理解平行系统中的这些过程，设计这样的途径，并开发新的抗菌药物。

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.