Time-resolved absorption and photothermal measurements with sensory rhodopsin I from Halobacterium salinarum.
An expansion accompanying the formation of the first intermediate in the photocycle of transducer-free sensory rhodopsin I (SRI) was determined by means of time-resolved laser-induced optoacoustic spectroscopy. For the native protein (SRI-WT), the absolute value of the expansion is approximately 5.5 mL and for the mutant SRI-D76N, approximately 1.5 mL per mol of phototransformed species (in 0.5 M NaCl), calculated by using the formation quantum yield for the first intermediate (S610) of Phi610 = 0.4 +/- 0.05 for SRI-WT and 0.5 +/- 0.05 for SRI-D76N, measured by laser-induced optoacoustic spectroscopy and by laser flash photolysis. The similarity in Phi610 and in the determined value of the energy level of S610, E610 = (142 +/- 12) kJ/mol for SRI-WT and SRI-D76N indicates that Asp76 is not directly involved in the first step of the phototransformation. The increase with pH of the magnitude of the structural volume change for the formation of S610 in SRI-WT and in SRI-D76N upon excitation with 580 nm indicates also that amino acids other than Asp76, and other than those related to the Schiff base, are involved in the process. The difference in structural volume changes as well as differences in the activation parameters for the S610 decay should be attributed to differences in the rigidity of the cavity surrounding the chromophore. Except for the decay of the first intermediate, which is faster than in the SRI-transducer complex, the rate constants of the photocycle for transducer-free SRI in detergent suspension are strongly retarded with respect to wild-type membranes (this comparison should be done with great care because the preparation of both samples is very different).
Losi
A
A
Max-Planck-Institut für Strahlenchemie, Postfach 101365, D-45413 Mülheim an der Ruhr, Germany.
Braslavsky
S E
SE
Gärtner
W
W
Spudich
J L
JL
D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics; D055592:Biophysical Phenomena; D001703:Biophysics; D002151:Calorimetry; D003902:Detergents; D019617:Halobacterium salinarum / Q000737:chemistry* / Q000235:genetics; D025602:Halorhodopsins; D006863:Hydrogen-Ion Concentration; D010777:Photochemistry; D010782:Photolysis; D017354:Point Mutation; D025603:Sensory Rhodopsins; D012995:Solubility; D013053:Spectrophotometry; D013816:Thermodynamics
D003902:Detergents; D025602:Halorhodopsins; C061497:SRI protein, Halobacterium; D025603:Sensory Rhodopsins; D001436:Bacteriorhodopsins