Abstract
The haloarchaeal phototaxis receptor sensory rhodopsin I (SRI) in complex with its transducer HtrI delivers an attractant signal from excitation with an orange photon and a repellent signal from a second near-UV photon excitation. Using a proteoliposome system with purified SRI in complex with its transducer HtrI, we identified by site-directed fluorescence labeling a site (Ser 155) on SRI that is conformationally active in signal relay to HtrI. Using site-directed spin labeling of Ser 155Cys with a nitroxide side chain, we detected a change in conformation following one-photon excitation such that the spin probe exhibits a splitting of the outer hyperfine extrema ( 2 A ′ zz ) significantly smaller than that of the electron paramagnetic resonance spectrum in the dark state. The dark conformations of five mutant complexes that do not discriminate between orange and near-UV excitation show shifts to lower or higher 2 A ′ zz values correlated with the alterations in their motility behavior to one- and two-photon stimuli. These data are interpreted in terms of a model in which the dark complex is populated by two conformers in the wild type, one that inhibits the CheA kinase ( A) and the other that activates it ( R), shifted in the dark by mutations and shifted in the wild-type SRI-HtrI complex in opposite directions by one-photon and two-photon reactions.
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