The three dimensional structure of the proteins involved in the phosphoenolpyruvate: sugar phosphotransferase system (PTS) will be determined by x-ray crystallography. This complex multi-enzyme system is one of the few transport systems whose components, including some of the membrane bound ones, have been purified. The crystallography will complement the molecular biology and biochemical aspects of a collaborative effect to study the mechanism by which bacteria employ a protein phosphoryl transfer chain to regulate sugar transport across the membrane. The structure determination of the various proteins will facilitate the analysis of the nature of regulation by protein phosphorylation, and the conformational transition that accompanies such processes. Since the PTS is unique to bacteria, the information obtained may pertain to our ability to take advantage of the PTS therapeutically. At this state, the focus is on the soluble proteins. Similar approaches will be used to obtain information about the phosphorylated structures of the other PTS proteins. Structure determination will be done either by the Multiple Isomorphous Replacement or by the Molecular Replacement methods, depending on the problem. For better accuracy, the determined structures will be refined to the highest resolution possible. Once structures become available, mutants that probe the protein-protein and protein-substrate interactions will be designed and analyzed biochemically, and selected interesting mutants will be crystallized and their structure determined.
