Export of Proteins in Escherichia coli

描述（由申请人提供）：我们的目的是阐明大肠杆菌中蛋白质输出的机制细节，重点是所涉及的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转换。蛋白质定位需要多肽跨生物膜转移，是所有生物体必需的普遍存在的过程。在E.大肠杆菌，在从单细胞生物到哺乳动物的所有三个生命界中高度保守。除了在几乎所有情况下通过膜的途径之外，无论该过程发生在原核生物还是真核生物中，伴侣都参与了早期阶段。我们建议开发一个分子描述的事件发生在出口，包括结合的伴侣SecB携带的前体多肽SecA，ATP酶电机的translocon，前体从SecB到SecA内的复杂和translsocation的前体多肽通过SecYEG translocon驱动SecA。我们将描绘SecA上的每个结合伙伴，SecB，前体和SecYEG的结合界面，并且我们将提供前体从SecB到SecA的动态转移过程中发生的构象变化的描述，以及参与SecYEG通道门控和开放的构象变化。为了实现这些目标，我们将使用一个组合的方法，范围从体外易位测定生物物理技术，如电子顺磁共振（EPR）光谱，这将使我们能够从一个一般性的相互作用的描述进行分子描述的决议的氨酰基侧链和多肽骨架结构。 蛋白质定位是一个对所有生物体都至关重要的过程。SecYEG复合物及其同系物是提供蛋白质转运通道的机器的普遍存在的组分。因此，我们通过研究细菌输出所学到的知识将适用于从细菌到人类的所有细胞中的现象。

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.