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.
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