title: Steric interaction between the 9-methyl group of the retinal and tryptophan 182 controls 13-cis to all-trans reisomerization and proton uptake in the bacteriorhodopsin photocycle.
abstract: The hypothesis was tested whether in bacteriorhodopsin (BR) the reduction of the steric interaction between the 9-methyl group of the chromophore all-trans-retinal and the tryptophan at position 182 causes the same changes as observed in the photocycle of 9-demethyl-BR. For this, the photocycle of the mutant W182F was investigated by time-resolved UV-vis and pH measurements and by static and time-resolved FT-IR difference spectroscopy. We found that the second half of the photocycle was similarly distorted in the two modified systems: based on the amide-I band, the protonation state of D96, and the kinetics of proton uptake, four N intermediates could be identified, the last one having a lifetime of several seconds; no O intermediate could be detected; the proton uptake showed a pronounced biphasic time course; and the pKa of group(s) on the cytoplasmic side in N was reduced from 11 in wild type BR to around 7.5. In contrast to 9-demethyl-BR, in the W182F mutant the first part of the photocycle does not drastically deviate from that of wild type BR. The results demonstrate the importance of the steric interaction between W182 and the 9-methyl group of the retinal in providing tight coupling between chromophore isomerization and the late proton transfer steps.
authors.affiliation: Institut für Biophysik und Strahlenbiologie, Albert-Ludwigs-Universität, Freiburg, Germany.
authors.forename: O
authors.initials: O
authors.lastname: Weidlich
authors.forename: B
authors.initials: B
authors.lastname: Schalt
authors.forename: N
authors.initials: N
authors.lastname: Friedman
authors.forename: M
authors.initials: M
authors.lastname: Sheves
authors.forename: J K
authors.initials: JK
authors.lastname: Lanyi
authors.forename: L S
authors.initials: LS
authors.lastname: Brown
authors.forename: F
authors.initials: F
authors.lastname: Siebert
mesh_terms: D006217:Halobacterium / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D007700:Kinetics; D010777:Photochemistry; D011522:Protons; D012172:Retinaldehyde / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D011522:Protons; D014364:Tryptophan; D012172:Retinaldehyde