Synthesis of a stable analog of the phosphorylated form of CheY: phosphono-CheY.
The chemical modification of a cysteinyl residue of D57C CheY by the addition of a phosphonomethyl group, (HO)(2)P(O)-CH(2)-, is described. This modification produces a nonlabile analog of an aspartyl phosphate residue in the active form of CheY. The chemically modified protein, phosphono-CheY, is suitable for structural and functional studies. An extensive discussion of the synthetic methodology and purification strategy is presented. A detailed protocol is given.
Halkides
Christopher J
CJ
Department of Chemistry and Biochemistry, University of North Carolina, Wilmington, North Carolina, USA.
Bottone
Cory J
CJ
Casper
Eric S
ES
Haas
R Matthew
RM
McAdams
Kenneth
K
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019921:Biotinylation; D002851:Chromatography, High Pressure Liquid; D004228:Dithionitrobenzoic Acid; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000072236:Methyl-Accepting Chemotaxis Proteins; D063065:Organophosphonates / Q000737:chemistry*; D010750:Phosphoproteins / Q000737:chemistry; D010766:Phosphorylation
D001426:Bacterial Proteins; D008565:Membrane Proteins; D000072236:Methyl-Accepting Chemotaxis Proteins; D063065:Organophosphonates; D010750:Phosphoproteins; C005149:phosphonoproteins; D004228:Dithionitrobenzoic Acid