Femtosecond and Picosecond Dynamics of Recombinant Bacteriorhodopsin Primary Reactions Compared to the Native Protein in Trimeric and Monomeric Forms.

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