This proposal addresses bacterial signal transduction at the molecular level, meaning how bacteria sense and respond to changes in their environment. More specifically the proposed research focusses on chemotaxis, the movement of bacteria up or down a chemical concentration gradient. Bacterial signal transduction depends upon a family of proteins known as response regulators, of which CheY, the response regulator in chemotaxis, is the most well studied. CheY is a signalling protein with an inactive state and a short-lived active state. In its active state CheY interacts with two proteins, CheZ and FliM. Until the recent creation of long-lived analogs of the active state, it had been difficult to study the active state of CheY and other response regulators. Response regulators are a logical target for drug design because mammals do not possess this family of proteins. Specifically, drugs that disrupt chemotaxis in pathogenic bacteria might thwart their pathogenicity. ? ? This research will use X-ray crystallography to solve the structures of CheY proteins and will use fluorescence quenching to determine the dissociation constants of peptides derived from CheZ and FliM. Along with the known phenotypes of mutants of CheY, these data will allow us to define which portions of the structure affect the function of CheY. Specifically, this research will test the hypothesis that residue Tyr106 is part of the signalling surface by using the Thr87Ile mutant of CheY. Signalling in this mutant is thought to be impaired because Ile87 forces the rotameric position of Tyr106 into its nonsignalling state. In addition, we will study the Lys109Arg mutant of CheY, which is also impaired in its ability to signal, by the same means. We will co-crystallize active CheY with a peptide derived from CheZ to determine whether or not CheZ promotes the phosphatase activity of CheY by inserting a residue directly into the active site of CheY.
| Lookadoo, Daniel B; Beyersdorf, Matthew S; Halkides, Christopher J (2018) Synthesis of a Stable Analog of the Phosphorylated Form of CheY: Phosphono-CheY. Methods Mol Biol 1729:337-343 |
| Sircar, Ria; Borbat, Peter P; Lynch, Michael J et al. (2015) Assembly states of FliM and FliG within the flagellar switch complex. J Mol Biol 427:867-886 |
| McAdams, Kenneth; Casper, Eric S; Matthew Haas, R et al. (2008) The structures of T87I phosphono-CheY and T87I/Y106W phosphono-CheY help to explain their binding affinities to the FliM and CheZ peptides. Arch Biochem Biophys 479:105-13 |
| Halkides, Christopher J; Bottone, Cory J; Casper, Eric S et al. (2007) Synthesis of a stable analog of the phosphorylated form of CheY: phosphono-CheY. Methods Enzymol 422:338-51 |
