title: Synthesis and characterization of a stable analog of the phosphorylated form of the chemotaxis protein CheY.
abstract: The bacterial chemotaxis protein CheY is activated in vivo by the covalent phosphorylation of a single aspartate residue at position 57. However, this phosphate linkage is unstable (t1/2 approximately 20 s at room temperature), thereby precluding many biochemical analyses. Here we present a synthetic scheme to prepare an analog of CheY-phosphate (Che Y-P) with chemical stability of the phosphate linkage enhanced by several orders of magnitude relative to the native protein. Starting with CheY D57C, a site-specific mutant of CheY with a unique cysteine residue in place of the aspartate at position 57, two sequential disulfide exchange reactions were performed to form the final product 'CheY D57C-SPO3' with a thiophosphate moiety covalently bonded to the protein in a disulfide linkage. Mass spectral analysis showed that the desired analog was present at 70-80% of the total protein. The disulfide linkage had a t1/2 of 8 days at 4 degrees C. Biochemical characterization of CheY D57C-SPO3 included assessment of conformational properties using tryptophan fluorescence, evaluation of metal binding properties and measurement of binding interactions with the chemotaxis proteins CheZ and FliM. Despite possessing a phosphoryl group at a nearly identical location as native CheY-phosphate, the analog was unable to emulate CheY-phosphate function, thereby supporting the idea that there are very precise geometric requirements for successful CheY activation.
authors.affiliation: Department of Microbiology and Immunology, University of North Carolina, Chapel Hill 27599-7290, USA.
authors.forename: R E
authors.initials: RE
authors.lastname: Silversmith
authors.forename: R B
authors.initials: RB
authors.lastname: Bourret
mesh_terms: D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002633:Chemotaxis; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D029968:Escherichia coli Proteins; D005453:Fluorescence; D008565:Membrane Proteins / Q000138:chemical synthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008670:Metals / Q000737:chemistry / Q000378:metabolism; D000072236:Methyl-Accepting Chemotaxis Proteins; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D008670:Metals; D000072236:Methyl-Accepting Chemotaxis Proteins; D010710:Phosphates; C000607606:cheY protein, E coli; C066821:FliM protein, Bacteria; D001224:Aspartic Acid; D003545:Cysteine; C035638:thiophosphoric acid