Export of Proteins in Escherichia coli

描述(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，重点是所涉及的蛋白质组分之间的结合作用所引起的功能状态之间的构象转换。蛋白质定位是一个无处不在的过程，它需要通过生物膜转移多肽，对所有活着的生物体来说都是必不可少的。通过膜的途径是由大肠杆菌中的异源三聚体复合体SecYEG提供的，在从单细胞生物体到哺乳动物的所有三个生命王国中都高度保守。除了几乎所有情况下通过膜的途径，无论这个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结合，转运子的ATPase马达，前体在复合体中从SecB进入SecA，以及前体多肽通过SecA驱动的SecYEG转位。我们将描述其每个结合伙伴SecB、前体和SecYEG在SecA上的结合界面，并描述前体从SecB到SecA动态转移过程中发生的构象变化，以及涉及SecYEG通道的门控和开放的构象变化。为了实现这些目标，我们将使用一系列方法的组合，从体外易位分析到生物物理技术，如电子顺磁共振(EPR)光谱，这将使我们能够从一般的相互作用描述转向对氨基酰基侧链和多肽主干结构的分子描述。 蛋白质定位是一个对所有生物都至关重要的过程。SecYEG复合体及其同系物是为蛋白质运输提供通道的机器中无处不在的组件。因此，我们通过研究细菌输出所学到的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.