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)

摘要

Phamonis 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 Schiffbase.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.