Heterogeneity effects in the binding of all-trans retinal to bacterio-opsin.

The special trimeric structure of bacteriorhodopsin (bR) in the purple membrane of Halobacterium salinarum, and especially, the still controversial question as to whether the three protein components are structurally and functionally identical, have been subject to considerable work. In the present work, the problem is approached by studying the reconstitution reaction of the bR apo-protein with all-trans retinal, paying special attention to the effects of the apo-protein/retinal (P:R) ratio. The basic observation is that at high P:R values, the reconstitution reaction proceeds via two distinct, fast and slow, pathways associated with two different pre-pigment precursors absorbing at 430 nm (P(430)) and 400 nm (P(400)), respectively. These two reactions, exhibiting 2:1 (P(430)/P(400)) amplitude ratios, are markedly affected by the P:R value. The principal feature is the acceleration of the P(400) --> bR transition at low P:R ratios. The data are interpreted in terms of a scheme in which the added retinal first occupies two protein retinal traps, R(1) and R(2), from which it is transferred to two spectroscopically distinct binding sites corresponding to the two pre-pigments, P(430) and P(400), respectively. Two noncovalently bound retinal molecules occupy two P(430) sites of the bR trimer, while one (P(400)) occupies the third. Binding is completed by generating the retinal-protein covalent bond. Analogous experiments were also carried out with an aromatic bR chromophore and with the D85N bR mutant. The accumulated data clearly point out the heterogeneity of the binding reaction intermediates, in which two are clearly distinct from the third. However, CD spectroscopy strongly suggests that even the two P(430) sites are not structurally identical. The heterogeneity of the P intermediates in the binding reaction can be accounted for, either by being induced by cooperativity or by an intrinsic heterogeneity that is already present in the apoprotein. The question as to whether the final reconstituted pigment, as well as native bR, are nonhomogeneous should be the subject of future studies.

Light-induced charge redistribution in the retinal chromophore is required for initiating the bacteriorhodopsin photocycle.

Bacteriorhodopsin's photocycle is initiated by the retinal chromophore light absorption. It has usually been assumed that light primarily isomerizes a retinal double bond which in turn induces protein conformational alterations and biological activity. We have studied several artificial pigments derived from retinal analogues tailored to substantially reduce the light-induced chromophore polarization. The lack of chromophore polarization was reflected in an undetectable second harmonic generation (SHG) signal. It was revealed that these artificial pigments did not exhibit any detectable light-induced photocycle nor light acceleration of the hydroxylamine-bleaching reaction. We suggest that light-induced retinal polarization triggers protein polarization which controls the course of the isomerization reaction by determining the relative efficiency of forward versus back-branching processes.

Following evolution of bacteriorhodopsin in its reactive excited state via stimulated emission pumping.

New information concerning the photochemical dynamics of bacteriorhodopsin (BR) is obtained by impulsively stimulating emission from the reactive fluorescent state. Depletion of the excited-state fluorescence leads to an equal reduction in production of later photoproducts. Accordingly, chromophores which are forced back to the ground state via emission do not continue on in the photocycle, conclusively demonstrating that the fluorescent state is a photocycle intermediate. The insensitivity of depletion dynamics to the "dump" pulse timing, throughout the fluorescent states lifetime, and the biological inactivity of the dumped population suggest that the fluorescent-state structure is constant, well-defined, and significantly different than that where crossing to the ground state takes place naturally. In conjunction with conclusions from comparing the photophysics of BR with those of synthetic analogues containing "locked" retinals, present results show that large-amplitude torsion around C13=C14 is required to go between the above structures.

Photoreduction of bacteriorhodopsin Schiff base at low humidity. A study with C13=C14 nonisomerizable artificial pigments.

The retinal protonated Schiff base of bacteriorhodopsin is photoreactive to reducing agents such as NaBH4. In the present work we have studied the effect of different protein hydration levels on the photoreductive reaction, as well as the consequences of preventing isomerization around the critical C13=C14 retinal double bond. It was revealed that the rate of light-induced NaBH4 reaction can be fitted to three phases, between 100 and 87%, from 87 to 35% and below 35% relative humidities (r.h.). The three phases are attributed to three protein regions characterized by different water affinities. Furthermore, it is shown that the PSB reduction reaction is light catalyzed even in artificial pigments derived from retinal analogs, in which isomerization around the C13=C14 double bond is prevented. It is suggested that the protein experiences light-induced conformational alterations that are not associated with C13=C14 double bond isomerization. In the 13-cis locked pigment the rate of reduction reaction is affected by r.h. levels only below 35%. The relatively low r.h. required for withdrawing water from the protein is attributed to the increased protein-water affinity in this specific pigment.

Light-induced hydrolysis and rebinding of nonisomerizable bacteriorhodopsin pigment.

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-cis locked bR pigment (bR5.13; lambda(max) = 550 nm) in which isomerization around the critical C13==C14 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 (lambda(max) = 450 nm) and H430 (lambda(max) = 430 nm), which are characterized by a 13-cis 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.