Transient movement of helix F revealed by photo-induced inactivation by reaction of a bulky SH-reagent to cysteine-introduced pharaonis phoborhodopsin (sensory rhodopsin II).
Pharaonis phoborhodopsin (ppR) is a photosensor of negative phototaxis in Natronomonas (Natronobacterium) pharaonis, an alkalophilic halophile. This protein has seven transmembrane helices into which a chromophore, all-trans retinal, binds to a specific lysine residue (located in helix G)via a protonated Schiff base. Various mutants were engineered to have a single cysteine in the F-helix. In the presence of a bulky fluorescent SH-reagent, MIANS, (2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid, illumination decreased the photoreactivity or flash-yield (absorbance deflection immediately after the flash) of the L163C ppR mutant (in which Leu-163 was replaced with Cys) without changing the photocycling rate. The fluorescence of the isolated protein increased with increasing illumination. These observations suggest that during photocycling, the space around Cys-163 in the F-helix might open, permitting reaction with the relatively large molecule. This reaction occurred only at the M-state and not at the O-state. The implications are discussed.
Yoshida
Hideaki
H
Laboratory of Biophysical Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan.
Sudo
Yuki
Y
Shimono
Kazumi
K
Iwamoto
Masayuki
M
Kamo
Naoki
N
D001426:Bacterial Proteins / Q000737:chemistry / Q000187:drug effects / Q000528:radiation effects; D006217:Halobacterium; D025602:Halorhodopsins / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D008027:Light; D008958:Models, Molecular; D016296:Mutagenesis; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000528:radiation effects; D025603:Sensory Rhodopsins / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D013438:Sulfhydryl Compounds / Q000494:pharmacology*
D001426:Bacterial Proteins; D025602:Halorhodopsins; D011994:Recombinant Proteins; D025603:Sensory Rhodopsins; D013438:Sulfhydryl Compounds; C061508:sensory rhodopsin II protein, archaeal