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组分的相互作用。特定的、新合成的多肽在生物膜上的转移是一个普遍存在的过程，这对活细胞是必不可少的。无论这个过程发生在真核生物还是原核生物中，在几乎所有的情况下，都涉及分子伴侣。伴侣是一类蛋白质，基于配体处于非本地状态这一事实，它们显示出非凡的识别和结合多肽的能力。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。这些实验旨在纳入从x射线晶体结构中获得的见解。将采用多种方法相结合的方法来定位非本地多肽的结合部位，定义所进行的接触，并描述所发生的构象变化。有关分子机制的结论将在体外和体内得到证实，方法是使用定点突变引入特定的变化，预计会消除伴侣活性的结合和丧失。 除了分子伴侣的参与，包括蛋白质输出在内的许多生物现象的一个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成分(如核苷酸和膜小泡)的情况下，提供作为激活开关的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主干的组织水平和侧链之间的接触所涉及的事件的分子描述。拟议的项目提供了各种生化和生物物理方法之间的平衡，这些方法相辅相成，相互加强。基于对纯化蛋白的体外研究得出的结论将在体内得到证实。通过整合通过不同方法获得的数据，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.