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Application of Fluorescence Resonance Energy Transfer (FRET) to Investigation of Light‐Induced Conformational Changes of the Phoborhodopsin/Transducer Complex †
Author(s) -
Taniguchi Yukinori,
Ikehara Tatsuya,
Kamo Naoki,
Watanabe Yasutaka,
Yamasaki Hiroshi,
Toyoshima Yoshinori
Publication year - 2007
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1562/2006-06-15-ra-922
Subject(s) - förster resonance energy transfer , fluorescence , chemistry , acceptor , biophysics , rhodopsin , photochemistry , optics , biology , biochemistry , physics , retinal , condensed matter physics
The photoreceptor phoborhodopsin ( p pR; also called sensory rhodopsin II) forms a complex with its cognate the Halobacterial transducer II ( p HtrII) in the membrane, through which changes in the environmental light conditions are transmitted to the cytoplasm in Natronomonas pharaonis to evoke negative phototaxis. We have applied a fluorescence resonance energy transfer (FRET)‐based method for investigation of the light‐induced conformational changes of the p pR/ p HtrII complex. Several far‐red dyes were examined as possible fluorescence donors or acceptors because of the absence of the spectral overlap of these dyes with all the photointermediates of p pR. The flash‐induced changes of distances between the donor and an acceptor linked to cysteine residues which were genetically introduced at given positions in p HtrII(1–159) and p pR were determined from FRET efficiency changes. The dye‐labeled complex was studied as solubilized in 0.1% n ‐dodecyl‐β‐ d ‐maltoside (DDM). The FRET‐derived changes in distances from V78 and A79 in p HtrII to V185 in p pR were consistent with the crystal structure data (Moukhametzianov, R. et al. [2006] Nature , 440, 115–119). The distance from D102 in p HtrII linker region to V185 in p pR increased by 0.33 Å upon the flash excitation. These changes arose within 70 ms (the dead time of instrument) and decayed with a rate of 1.1 ± 0.2 s. Thus, sub‐angstrom‐scale distance changes in the p pR/ p HtrII complex were detected with this FRET‐based method using far‐red fluorescent dyes; this method should be a valuable tool to investigate conformation changes in the transducer, in particular its dynamics.