The long-term objective of this project is to understand the mechanism of membrane transport as illustrated by operation of the """"""""secondary"""""""" carriers, a class of transport proteins that function in both nutrient accumulation and waste product excretion, in all cells. Defects in carrier-mediated events are commonly associated with malabsorption syndromes, and the analysis of these proteins will provide clues as to the proper treatment and cure in disease states. As model carriers, this project will use anion exchange proteins. Most work will focus on UhpT, a 46 kD protein mediating the exchange of inorganic and/or organic phosphates in Escherichia coli, UhpT is well-defined by biochemical and genetic work. It may also be of value to study OxLT, the oxalate-formate exchange protein of Oxalobacter formigenes; OxLT is unusually stable in its solubilized form, and this may be important to structural studies. Interdependent experimental approaches will address central features of transport. Thus, arginine and/or lysine residues appear to play important roles in UhpT and related systems, and to confirm and extend these findings, oligonucleotide-directed mutagenesis will replace these and additional residues conserved among UhpT and its relatives, with added mutagenesis of other residues located by chemical modification. To further identify residues of functional significance, we will select mutants of substrate-specificity. Along with the sequences of new relatives of UhpT, particularly those in Salmonella typhimurium, this approach should reveal critical substrate-binding domains within UhpT. In complementary work, the Uhpt gene will be subdivided, its fragments will be simultaneously or separately expressed in E. coli, and the functional products will be purified to characterize a minimal transport structure. For this and for the parental material, subsequent studies with cross-linking agents will then indicate points of protein-protein contact. Finally, purification methods will be optimized to yield large amounts of high quality material for 2D and 3D structural work. Such genetic, and biochemical studies should reveal the essential features of overall protein structure and identify domains involved in substrate binding and transport.
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