Export of Proteins in Escherichia coli

描述（由申请人提供）：我们的目标是阐明大肠杆菌中蛋白质输出的机制细节，重点是由相关蛋白质组分之间的结合相互作用诱导的功能状态之间的构象切换。蛋白质定位需要多肽在生物膜上的转移，这是一个普遍存在的过程，对所有生物体都是必不可少的。大肠杆菌中的异源三聚体复合物SecYEG提供了通过膜的途径，在从单细胞生物到哺乳动物的所有三个生命领域中都是高度保守的。除了在几乎所有情况下通过膜的途径外，无论该过程发生在原核生物还是真核生物中，伴侣蛋白都参与了早期阶段。我们建议对出口中发生的事件进行分子描述，包括携带前体多肽的伴侣SecB与SecA的结合、转座子的atp酶马达、前体在复合物内从SecB到SecA的传递以及由SecA驱动的前体多肽通过SecYEG转座子的易位。我们将描述SecA上每个结合伙伴（SecB、前体和SecYEG）的结合界面，并描述前体从SecB到SecA的动态转移过程中发生的构象变化，以及与SecYEG通道的门控和打开有关的构象变化。为了实现这些目标，我们将结合使用各种方法，从体外易位测定到生物物理技术，如电子顺磁共振（EPR）光谱，这将使我们能够从相互作用的一般描述到氨基酰基侧链和多肽主链结构的分子描述。

DESCRIPTION (provided by applicant): We aim to elucidate the mechanistic details of protein export in Escherichia coli with emphasis on conformational switches between functional states induced by binding interactions among the protein components involved. Protein localization, which requires transfer of polypeptides across biological membranes, is a ubiquitous process essential to all living organisms. The pathway through the membrane, provided by a heterotrimeric complex SecYEG in E. coli, is highly conserved in all three kingdoms of life going from single cell organisms to mammals. In addition to a pathway through the membrane in almost all cases, whether the process occurs in prokaryotes or eukaryotes, chaperones are involved in the early stages. We propose to develop a molecular description of the events that occur in export including the binding of the chaperone SecB carrying a precursor polypeptide to SecA, the ATPase motor of the translocon, the passage of the precursor from SecB to SecA within the complex and tranlsocation of the precursor polypeptide through the SecYEG translocon driven by SecA. We shall delineate the binding interfaces on SecA for each of its binding partners, SecB, precursors and SecYEG, and we shall provide a description of conformational changes that occur during the dynamic transfer of the precursor from SecB to SecA as well as the conformational changes that are involved in gating and opening of the SecYEG channel. To achieve these goals we shall use a combination of approaches ranging from in vitro translocation assays to biophysical techniques such as electron paramagnetic resonance (EPR) spectroscopy that will allow us to proceed from a general description of interactions to a molecular description at the resolution of aminoacyl side chains and polypeptide backbone structure. Protein localization is a process that is essential for all living organisms. The SecYEG complex and its homologs are the ubiquitous component of the machines that provide the channel for protein transport. Therefore, what we learn by studying bacterial export will be applicable to the phenomenon in all cells, from bacteria to humans.