Export of Proteins in Escherichia coli

描述（由申请方提供）：目的是阐明大肠杆菌中蛋白质输出的机制，重点是途径中蛋白质组分的相互作用。 新合成的特异性多肽跨生物膜的转运是一个普遍存在的过程，这对于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。 伴侣蛋白是一类基于配体处于非天然状态的事实而显示出识别和结合多肽的显著能力的蛋白质家族。 研究人员的目标是通过研究伴侣SecB及其配体之间的相互作用，进一步探索这种序列独立识别的分子基础。 这些实验的目的是结合从X射线晶体结构中获得的见解。 一个组合的方法将被应用到定位非天然多肽的结合位点，以确定接触和描绘构象发生的变化。 关于分子机制的结论将在体外和体内通过使用定点诱变引入预测消除结合和伴侣活性丧失的特异性变化来证实。 除了分子伴侣的参与外，包括蛋白质输出在内的许多生物现象的共同主题是活性状态的构象转换。 研究人员将通过检查SecB，多肽配体和SecA之间的相互作用，在存在和不存在其他组分（如核苷酸和膜囊泡）的情况下，提供构象变化的分子描述。 申请人将采用广泛的技术从构象变化的一般描述转移到多肽骨架组织水平和侧链之间接触所涉及事件的分子描述。 拟议的项目提供了各种生物化学和生物物理方法之间的平衡，相互补充和加强。 基于体外纯化蛋白工作的结论将在体内得到证实。 正是从通过不同方法获得的数据的整合中，调查人员将学到最多的东西。

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.