Export of Proteins in Escherichia coli

描述（由申请人提供）：目的是阐明大肠杆菌中蛋白质输出的机制，重点是该途径中蛋白质成分的相互作用。特定的、新合成的多肽在生物膜上的易位是一个普遍存在的过程，这对活细胞是必不可少的。无论这一过程发生在真核生物还是原核生物中，几乎所有情况下都涉及分子伴侣。伴侣蛋白是一类蛋白质，基于配体处于非天然状态的事实，显示出识别和结合多肽的非凡能力。研究人员的目标是通过研究伴侣SecB与其配体之间的相互作用，进一步探索这种序列无关识别的分子基础。设计这些实验是为了结合从x射线晶体结构中获得的见解。将应用多种方法来定位非天然多肽的结合位点，定义所形成的接触并描述发生的构象变化。关于分子机制的结论将在体外和体内得到证实，通过使用位点定向诱变来引入预测的消除结合和伴侣活性丧失的特异性变化。

DESCRIPTION (provided by applicant): The objective is to elucidate the mechanism of protein export in Escherichia coli with emphasis on the interactions of the protein components of the pathway. Translocation of specific, newly synthesized polypeptides across biological membranes is a ubiquitous process, which is essential for living cells. Whether the process occurs in eukaryotes or in prokaryotes in almost all cases molecular chaperones are involved. Chaperones are a family of proteins that display the remarkable ability to recognize and bind polypeptides based on the fact that the ligands are in a nonnative state. The investigators aim to further explore the molecular basis of this sequence-independent recognition by studies of interactions between the chaperone SecB and its ligands. The experiments have been designed to incorporate insights obtained from the x-ray crystal structure. A combination of approaches will be applied to locate the binding site for nonnative polypeptides, to define contacts made and to delineate changes in conformation which occur. Conclusions regarding the molecular mechanism will be confirmed in vitro as well as in vivo by using site-directed mutagenesis to introduce specific changes predicted to eliminate binding and loss of the chaperone activity. In addition to participation of molecular chaperones, a theme common to many biological phenomena including protein export is that of conformational switching of active states. The investigators will provide a molecular description of changes in conformation that serve as activational switches by examining interactions among SecB, polypeptide ligands and SecA, in the presence and absence of other components such as nucleotides and membrane vesicles. The applicants will employ a wide range of techniques to move from a general description of changes in conformation to a molecular description of the events involved at the level of organization of the polypeptide backbone and contacts between side chains. The proposed projects provide a balance among a variety of biochemical and biophysical approaches that complement and reinforce one another. Conclusions that are based on work in vitro with purified proteins will be confirmed in vivo. It is from integration of data obtained through diverse approaches that the investigators will learn the most.