Femtosecond and Picosecond Dynamics of Recombinant Bacteriorhodopsin Primary Reactions Compared to the Native Protein...

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.