Genetic Analysis of Complex Membrane Protein Insertion

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The aim of this research is to provide a better understanding of how membrane proteins acquire their final structures. The experiments analyze two >E.> >coli> cytoplasmic membrane proteins of simple and complex topological structures, the >trs> serine chemoreceptor and >lac> permease. The research aims to help elucidate both how the transmembrane topological structures of these proteins are generated and how the topological structures fold in three dimensions into globular structures. This research tests new genetic strategies for elucidating the topological structures of cytoplasmic membrane proteins. It also aims to analyze nonfunctional mutants to identify the sequence features of transmembrane segments that enable them to direct the establishment of topology, and to identify any cellular components with which the transmembrane segments interact in this function. The analysis relies on the use of "sensor" proteins whose enzymatic activities reflect the normal cellular location of the membrane protein domain to which they are attached in hybrid proteins. The research analyzes how the topological structure of >lac> permease folds into a globular structure using mutants that are defective in the process. Such mutants will be identified as a subset of mutants unable to transport lactose, or constructed >in> >vitro> by altering amino acid residues found to be conserved in evolution.