open access

https://hdl.handle.net/2115/33764

Application of fluorescence resonance energy transfer (FRET) to investigation of light-induced conformational changes of the phoborhodopsin/transducer complex

英語

2007-03

Blackwell Publishing

Photochemistry and Photobiology

83(2)

311-316

The photoreceptor phoborhodopsin (ppR; also called sensory rhodopsin II) forms a complex with its cognate the Halobacterial transducer II (pHtrII) 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 ppR/pHtrII 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 ppR. The flash-induced changes of distances between the donor and an acceptor linked to cysteine residues which were genetically introduced at given positions in pHtrII(1-159) and ppR 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 pHtrII to V185 in ppR were consistent with the crystal structure data [Moukhametzianov, R. et al. (2006) Nature, 440, 787-792]. The distance from D102 in pHtrII linker region to V185 in ppR increased by 0.33Å upon the flash excitation. These changes arose within 70ms (the dead time of instrument) and decayed with a rate of 1.1±0.2s. Thus, sub angstrom-scale distance changes in the ppR/pHtrII 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.

学術雑誌論文

Accepted Manuscript

Author Posting. © The Authors 2007 This is the author's version of the work. It is posted here for personal use, not for redistribution. The definitive version was published in Photochemistry and Photobiology, Volume 83, Issue 2, Page 311-316. http://dx.doi.org/10.1562/2006-06-15-RA-922

0031-8655

AA00773103