Binding of the Chemotaxis Response Regulator CheY to the Isolated, Intact Switch Complex of the Bacterial Flagellar Motor: LACK OF COOPERATIVITY *

摘要: Abstract In bacteria, the chemotactic signal is greatly amplified between chemotaxis receptors and flagellar motor. Escherichia coli, part of this amplification occurs at switch. However, it not known whether results from cooperativity CheY binding to switch or a post-binding step. To address question, we purified intact complex (constituting proteins FliG, FliM, FliN scaffolding protein FliF) in quantities sufficient for biochemical work used investigate cooperative. As negative control, complexes switchless basal bodies, formed FliF FliG similarly isolated. Using double-labeling centrifugation assays binding, found that binds isolated, phosphorylation-dependent manner. We observed no significant control body. The dissociation constant phosphorylated (CheY∼P) was 4.0 ± 1.1 μm, well line with published range CheY∼P concentrations which motor responsive. Furthermore, cooperative (Hill coefficient ≈ 1). This lack CheY∼P-switch cooperativity, taken together earlier vivo studies suggesting dependence rotational state on fraction occupied sites sigmoidal very steep (Bren, A., Eisenbach, M. (2001) J. Mol. Biol. 312, 699–709), indicates within switch, subsequent binding.