Transport is an essential and often rate limiting step in the entry of nutrients and ions into cells. However, despite many elegant biophysical studies on transport proteins, very little is known about the actual molecular mechanisms of transport. The ease of isolation and molecular characterization of proline transport mutants in Salmonella typhimurium makes this a good model system for studying the molecular mechanism of sodium-driven transport. We plan to isolate mutants of S. typhimurium defective for proline transport in order to identify the active site or proline permease. By selecting for mutants with altered substrate of counter-ion specificity, domains of the permease involved in substrate binding and translocation can be identified. DNA sequence analysis of the mutants will identify the amino acid residues that directly interact with the substrate and counter-ion. The kinetics of substrate binding and translocation will be measured for each mutant. The phenotype of mutants with specific amino acid substitutions may indicate the function of these amino acids in substrate translocation. Site directed mutagenesis of the residues implicated in transport will allow us to test the predicted role of specific amino acids in substrate translocation. In order to determine the position of the active site in the protein, the structure of proline permease will be predicted from the DNA sequence of the putP gene and the topology of the protein in the membrane will be determined using gene fusions. Such genetic and biochemical analysis of proline permease may elucidate the structure and function of ion-driven active transport systems.
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