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The primary stages of the Exiguobacterium sibiricum rhodopsin (ESR) photocycle were investigated by femtosecond absorption laser spectroscopy in the spectral range of 400-900 nm with a time resolution of 25 fs. The dynamics of the ESR photoreaction were compared with the reactions of bacteriorhodopsin (bR) in purple membranes (bR PM ) and in recombinant form (bR rec ). The primary intermediates of the ESR photocycle were similar to intermediates I , J , and K in bacteriorhodopsin photoconversion. The CONTIN program was applied to analyze the characteristic times of the observed processes and to clarify the reaction scheme. A similar photoreaction pattern was observed for all studied retinal proteins, including two consecutive dynamic Stokes shift phases lasting ∼0.05 and ∼0.15 ps. The excited state decays through a femtosecond reactive pathway, leading to retinal isomerization and formation of product J , and a picosecond nonreactive pathway that leads only to the initial state. Retinal photoisomerization in ESR takes 0.69 ps, compared with 0.48 ps in bR PM and 0.74 ps in bR rec . The nonreactive excited state decay takes 5 ps in ESR and ∼3 ps in bR. We discuss the similarity of the primary reactions of ESR and other retinal proteins.

The supramolecular organization of the visual pigment rhodopsin in the photoreceptor membrane remains contentious. Specifically, whether this G protein-coupled receptor functions as a monomer or dimer remains unknown, as does the presence or absence of ordered packing of rhodopsin molecules in the photoreceptor membrane. Completely opposite opinions have been expressed on both issues. Herein, using small-angle neutron and X-ray scattering approaches, we performed a comparative analysis of the structural characteristics of the photoreceptor membrane samples in buffer, both in the outer segment of photoreceptor cells, and in the free photoreceptor disks. The average distance between the centers of two neighboring rhodopsin molecules was found to be ~5.8 nm in both cases. The results indicate an unusually high packing density of rhodopsin molecules in the photoreceptor membrane, but molecules appear to be randomly distributed in the membrane without any regular ordering., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Photochemical reaction dynamics of the primary events in recombinant bacteriorhodopsin (bR rec ) was studied by femtosecond laser absorption spectroscopy with 25-fs time resolution. bR rec was produced in an Escherichia coli expression system. Since bR rec was prepared in a DMPC-CHAPS micelle system in the monomeric form, its comparison with trimeric and monomeric forms of the native bacteriorhodopsin (bR trim and bR mon , respectively) was carried out. We found that bR rec intermediate I (excited state of bR) was formed in the range of 100 fs, as in the case of bR trim and bR mon . Further processes, namely the decay of the excited state I and the formation of intermediates J and K of bR rec , occurred more slowly compared to bR trim , but similarly to bR mon . The lifetime of intermediate I, judging from the signal of ΔA ESA (470-480 nm), was 0.68 ps (78%) and 4.4 ps (22%) for bR rec , 0.52 ps (73%) and 1.7 ps (27%) for bR mon , and 0.45 ps (90%) and 1.75 ps (10%) for bR trim . The formation time of intermediate K, judging from the signal of ΔA GSA (625-635 nm), was 13.5 ps for bR rec , 9.8 ps for bR mon , and 4.3 ps for bR trim . In addition, there was a decrease in the photoreaction efficiency of bR rec and bR mon as seen by a decrease in absorbance in the differential spectrum of the intermediate K by ~14%. Since photochemical properties of bR rec are similar to those of the monomeric form of the native protein, bR rec and its mutants can be considered as a basis for further studies of the mechanism of bacteriorhodopsin functioning.