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Biophys. J. BioFAST: First Published March 9, 2007. doi:10.1529/biophysj.106.101121
© 2007 by the Biophysical Society.

A more recent version of this article appeared on June 1, 2007.
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PHOTOBIOPHYSICS

Different Dark Conformations Function in Color-Sensitive Photosignaling by the Sensory Rhodopsin I-HtrI Complex

Jun Sasaki 1, Brian J. Phillips 1, Xinpu Chen 1, Ned Van Eps 2, Ah-Lim Tsai 1, Wayne L Hubbell 2 and John L Spudich 1*

1 University of Texas Medical School at Houston
2 University of California School of Medicine, Los Angeles

* To whom correspondence should be addressed. E-mail: john.l.spudich{at}uth.tmc.edu.

Submitted on November 20, 2006
Revised on December 26, 2006
Accepted on 18 January 2007


  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 (Ser155) on SRI which is conformationally active in signal relay to HtrI. Using site-directed spin labeling of Ser155Cys 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 (2Azz') 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 2Azz' 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 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 wildtype SRI-HtrI complex in opposite directions by one-photon and two-photon reactions.

Key Words: electron paramagnetic resonance (EPR) spectroscopy, phototaxis, signal transduction, site directed spin labeling (SDSL)




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Copyright © 2007 by the Biophysical Society.