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

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O. A. Smitienko^1*, O. V. Nekrasova^2,3, A. V. Kudriavtsev^1,3, M. A. Yakovleva¹, I. V. Shelaev⁴, F. E. Gostev⁴, D. A. Dolgikh^2,3,5, I. B. Kolchugina³, V. A. Nadtochenko⁴, M. P. Kirpichnikov^2,3, T. B. Feldman^1,3,5, and M. A. Ostrovsky^1,3

¹Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, 119334 Moscow, Russia; E-mail: djolia@gmail.com

²Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia

³Lomonosov Moscow State University, Biological Faculty, 119991 Moscow, Russia

⁴Semenov Institute of Chemical Physics, Russian Academy of Sciences, 119991 Moscow, Russia

⁵Pirogov Russian National Research Medical University, 1117997 Moscow, Russia

^* To whom correspondence should be addressed.

Received October 22, 2016; Revision received December 20, 2016

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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.

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KEY WORDS: bacteriorhodopsin, recombinant protein, primary reactions, femtosecond absorption laser spectroscopy

DOI: 10.1134/S0006297917040113

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