RESUMO
CheY is a response regulator in bacterial chemotaxis. Escherichia coli CheY mutants T87I and T87I/Y106W CheY are phosphorylatable on Asp57 but unable to generate clockwise rotation of the flagella. To understand this phenotype in terms of structure, stable analogs of the two CheY-P mutants were synthesized: T87I phosphono-CheY and T87I phosphono-CheY. Dissociation constants for peptides derived from flagellar motor protein FliM and phosphatase CheZ were determined for phosphono-CheY and the two mutants. The peptides bind phosphono-CheY almost as strongly as CheY-P; however, they do not bind T87I phosphono-CheY or T87I/Y106W phosphono-CheY, implying that the mutant proteins cannot bind FliM or CheZ tightly in vivo. The structures of T87I phosphono-CheY and T87I/Y106W phosphono-CheY were solved to resolutions of 1.8 and 2.4A, respectively. The increased bulk of I87 forces the side-chain of Y106 or W106, into a more solvent-accessible conformation, which occludes the peptide-binding site.
Assuntos
Substituição de Aminoácidos , Proteínas de Bactérias/química , Escherichia coli/química , Proteínas de Membrana/química , Mutação de Sentido Incorreto , Peptídeos/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/fisiologia , Quimiotaxia/fisiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Quimiotáticas Aceptoras de Metil , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica/fisiologia , Estrutura Quaternária de Proteína/fisiologia , Estrutura Terciária de Proteína/fisiologiaRESUMO
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.
