Activation of the phosphosignaling protein CheY. II. Analysis of activated mutants by 19F NMR and protein engineering.

摘要: The Escherichia coli CheY protein is activated by phosphorylation, and in turn alters flagellar rotation. To investigate the molecular mechanism of activation, an extensive collection mutant proteins was analyzed behavioral assays, vitro 19F NMR chemical shift measurements. Substitution a positively charged residue (Arg or Lys) place Asp13 activation site results even for mutants which cannot be phosphorylated. Thus phosphorylation plays indirect role mechanism. Lys109, proposed to act as conformational "switch" site, required either mutation. assay described preceding article (Drake, S. K., Bourret, R. B., Luck, L. A., Simon, M. I., Falke, J. (1993) Biol Chem. 268, 13081-13088) again used monitor six phenylalanine positions CheY, including one position probed vicinity Lys109. Mutations activate were observed perturb Lys109 probe, providing further evidence that directly involved activating change. Two striking contrasts between mutation phosphorylation. (i) Each generates relatively localized perturbation region, whereas triggers global structural (ii) region mutations not detected phosphorylated protein. These are consistent with two-step model docking switch.