Application of fluorescence resonance energy transfer (FRET) to investigation of light-induced conformational changes of the phoborhodopsin/transducer complex.
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-beta-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, 115-119). The distance from D102 in pHtrII linker region to V185 in ppR increased by 0.33 angstroms 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 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.
Taniguchi
Yukinori
Y
Nano-biotechnology Research Center, and Department of Bioscience, School of Science & Technology, Kwansei Gakuin University, Sanda, Hyogo, Japan. guchi@ksc.kwansei.ac.jp
Ikehara
Tatsuya
T
Kamo
Naoki
N
Watanabe
Yasutaka
Y
Yamasaki
Hiroshi
H
Toyoshima
Yoshinori
Y
D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects*; D031541:Fluorescence Resonance Energy Transfer; D006216:Halobacteriaceae / Q000737:chemistry / Q000235:genetics / Q000528:radiation effects; D025602:Halorhodopsins / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects*; D046912:Multiprotein Complexes; D010777:Photochemistry; D011487:Protein Conformation / Q000528:radiation effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000528:radiation effects; D025603:Sensory Rhodopsins / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects*
D019843:Archaeal Proteins; D025602:Halorhodopsins; C434451:HtrII protein, Natronobacterium pharaonis; D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D025603:Sensory Rhodopsins; C061508:sensory rhodopsin II protein, archaeal