The lifetimes of Pharaonis phoborhodopsin signaling states depend on the rates of proton transfers--effects of hydrostatic pressure and stopped flow experiments.
Pharaonis phoborhodopsin (ppR), a negative phototaxis receptor of Natronomonas pharaonis, undergoes photocycle similar to the light-driven proton pump bacteriorhodopsin (BR), but the turnover rate is much slower due to much longer lifetimes of the M and O intermediates. The M decay was shown to become as fast as it is in BR in the L40T/F86D mutant. We examined the effects of hydrostatic pressure on the decay of these intermediates. For BR, pressure decelerated M decay but slightly affected O decay. In contrast, with ppR and with its L40T/F86D mutant, pressure slightly affected M decay but accelerated O decay. Clearly, the pressure-dependent factors for M and O decay are different in BR and ppR. In order to examine the deprotonation of Asp75 in unphotolyzed ppR we performed stopped flow experiments. The pH jump-induced deprotonation of Asp75 occurred with 60 ms, which is at least 20 times slower than deprotonation of the equivalent Asp85 in BR and about 10-fold faster than the O decay of ppR. These data suggest that proton transfer is slowed not only in the cytoplasmic channel but also in the extracellular channel of ppR and that the light-induced structural changes in the O intermediate of ppR additionally decrease this rate.
Kikukawa
Takashi
T
Creative Research Initiative Sosei, Hokkaido University, Sapporo, Japan. kikukawa@cast.hokudai.ac.jp
Saha
Chabita K
CK
Balashov
Sergei P
SP
Imasheva
Eleonora S
ES
Zaslavsky
Dmitry
D
Gennis
Robert B
RB
Abe
Takayuki
T
Kamo
Naoki
N
D001224:Aspartic Acid / Q000032:analysis; D001436:Bacteriorhodopsins / Q000737:chemistry / Q000528:radiation effects; D025602:Halorhodopsins / Q000737:chemistry* / Q000528:radiation effects; D006874:Hydrostatic Pressure; D007700:Kinetics; D008027:Light; D019633:Natronobacterium / Q000737:chemistry* / Q000528:radiation effects; D010782:Photolysis; D011522:Protons; D025603:Sensory Rhodopsins / Q000737:chemistry* / Q000528:radiation effects; D013053:Spectrophotometry
D025602:Halorhodopsins; D011522:Protons; D025603:Sensory Rhodopsins; C061508:sensory rhodopsin II protein, archaeal; D001224:Aspartic Acid; D001436:Bacteriorhodopsins