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• Trans/13-cis isomerization is essential for both the photocy...

  Trans/13-cis isomerization is essential for both the photocycle and proton pumping of bacteriorhodopsin Biophysical Journal, Volume 47, Issue 4, 1 April 1985, Pages 509-512 C.H. Chang, R. Govindjee, T. Ebrey, K.A. Bagley, G. Dollinger, L. Eisenstein, J. Marque, H. Roder, J. Vittitow and J.M. Fang Abstract We studied an analogue of bacteriorhodopsin whose chromophore is based on all-trans retinal. A five-membered ring was built around the 13–14 double bond so as to prohibit trans to 13-cis isomerization. No light-induced photochemical changes were seen, other than those due to a small amount (approximately 5%) of unbleached bacteriorhodopsin remaining in the apomembrane used for regeneration. The techniques used included flash photolysis at room and liquid nitrogen temperatures and Fourier-transform infrared difference spectroscopy. When the trans-fixed pigment was incorporated into phospholipid vesicles, no evidence of light-initiated proton pumping could be found. The results indicate that trans to 13-cis isomerization is essential for the photochemical transformation and function of bacteriorhodopsin. Abstract | PDF (463 kb)

• The Hydroxylamine Reaction of Sensory Rhodopsin II: Light-In...

  The Hydroxylamine Reaction of Sensory Rhodopsin II: Light-Induced Conformational Alterations with C13C14 Nonisomerizable Pigment Biophysical Journal, Volume 89, Issue 4, 1 October 2005, Pages 2610-2617 U. Zadok, J.P. Klare, M. Engelhard and M. Sheves Abstract Sensory rhodopsin II, a repellent phototaxis receptor from (NpSRII), forms a complex with its cognate transducer (NpHtrII). In micelles the two proteins form a 1:1 heterodimer, whereas in membranes they assemble to a 2:2 complex. Similarly to other retinal proteins, sensory rhodopsin II undergoes a bleaching reaction with hydroxylamine in the dark which is markedly catalyzed by light. The reaction involves cleavage of the protonated Schiff base bond which covalently connects the retinal chromophore to the protein. The light acceleration reflects protein conformation alterations, at least in the retinal binding site, and thus allows for detection of these changes in various conditions. In this work we have followed the hydroxylamine reaction at different temperatures with and without the cognate transducer. We have found that light irradiation reduces the activation energy of the hydroxylamine reaction as well as the frequency factor. A similar effect was found previously for bacteriorhodopsin. The interaction with the transducer altered the light effect both in detergent and membranes. The transducer interaction decreased the apparent light effect on the energy of activation and the frequency factor in detergent but increased it in membranes. In addition, we have employed an artificial pigment derived from a retinal analog in which the critical CC double bond is locked by a rigid ring structure preventing its isomerization. We have observed light enhancement of the reaction rate and reduction of the energy of activation as well as the frequency factor, despite the fact that this pigment does not experience CC double bond isomerization. It is suggested that retinal excited state polarization caused by light absorption of the “locked” pigment polarizes the protein and triggers relatively long-lived protein conformational alterations. Abstract | Full Text | PDF (124 kb)

• Light-Induced Hydrolysis and Rebinding of Nonisomerizable Ba...

  Light-Induced Hydrolysis and Rebinding of Nonisomerizable Bacteriorhodopsin Pigment Biophysical Journal, Volume 82, Issue 5, 1 May 2002, Pages 2617-2626 Amir Aharoni, Michael Ottolenghi and Mordechai Sheves Abstract Bacteriorhodopsin (bR) is characterized by a retinal-protein protonated Schiff base covalent bond, which is stable for light absorption. We have revealed a light-induced protonated Schiff base hydrolysis reaction in a 13- locked bR pigment (bR5.13; =550nm) in which isomerization around the critical CC double bond is prevented by a rigid ring structure. The photohydrolysis reaction takes place without isomerization around any of the double bonds along the polyene chain and is indicative of protein conformational alterations probably due to light-induced polarization of the retinal chromophore. Two photointermediates are formed during the hydrolysis reaction, H450 (=450nm) and H430 (=430nm), which are characterized by a 13- configuration as analyzed by high-performance liquid chromatography. Upon blue light irradiation after the hydrolysis reaction, these intermediates rebind to the apomembrane to reform bR5.13. Irradiation of the H450 intermediate forms the original pigment, whereas irradiation of H430 at neutral pH results in a red shifted species (P580), which thermally decays back to bR5.13. Electron paramagnetic resonance (EPR) spectroscopy indicates that the cytoplasmic side of bR5.13 resembles the conformation of the N photointermediate of native bR. Furthermore, using osmotically active solutes, we have observed that the hydrolysis rate is dependent on water activity on the cytoplasmic side. Finally, we suggest that the hydrolysis reaction proceeds via the reversed pathway of the binding process and allows trapping a new intermediate, which is not accumulated in the binding process. Abstract | Full Text | PDF (168 kb)

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Abstract

We have measured the emission lifetime of bacteriorhodopsin at physiological temperatures to be 15 +/- 3 ps using a technique which employs a mode-locked dye laser, a sum frequency light gate, and a continuous flow system. We observe no concentration dependence of the lifetime over the range of 1.1 X 10(-4) M to 1.0 X 10(-5) M. We conclude that the emission which we observe comes from bacteriorhodopsin and not one of its photochemically produced intermediates, and that the emission cannot originate from the state into which light is absorbed.