Temperature Dependence of the Krokinobacter rhodopsin 2 Kinetics

Abstract

We investigated the temperature-dependent kinetics of the light-driven Na þ pump Krokinobacter rhodopsin 2 (KR2) at Na þ -pumping conditions. The recorded microsecond flash photolysis data were subjected to detailed global target analysis, employing Eyring constraints and spectral decomposition. The analysis resulted in the kinetic rates, the composition of the different photocycle equilibria, and the spectra of the involved photointermediates. Our results show that with the temperature increase (from 10 to 40 C), the overall photocycle duration is accelerated by a factor of 6, with the L-to-M transition exhibiting an impressive 40-fold increase. It follows from the analysis that in KR2 the chromophore and the protein scaffold are more kinetically decoupled than in other microbial rhodopsins. We link this effect to the rigidity of the retinal protein environment. This kinetic decoupling should be considered in future studies and could potentially be exploited for fine-tuning biotechnological applications.

A subgroup of light-driven sodium pumps with an additional Schiff base counterion

“…We also studied the photocycle kinetics of Er NaR on the ns to s timescale under various conditions using flash photolysis. At pH 8.0, the photocycle, as well as the impact of different sodium concentrations on the Er NaR, are similar to what was observed for KR2 1 , 28 , 29 . The K 1 -intermediate at the end of the ultrafast measurement (1.8 ns), remains present at the beginning of the flash photolysis time scale (450 ns), showing that K 1 is populated throughout the ns timescale and no photocycle intermediate is missed due to the experimental time gap (Fig.…”

“…K 1 decays within the early µs-range (lifetime distribution in the range of 5–10 µs) to form the K 2 -intermediate. This intermediate state is considered to be K-like because of the more pronounced red-shift compared to the known L-intermediates of other microbial rhodopsins 28 , 29 . The transition to the blue-shifted M-intermediate then occurs within ~1 ms.…”

A novel subgroup of light-driven sodium pumps with an additional Schiff base counterion

“…We also studied the photocycle kinetics of Er NaR on the ns to s timescale under various conditions using flash photolysis. At pH 8.0, the photocycle, as well as the impact of different sodium concentrations on the Er NaR, are similar to what was observed for KR2 1,30,31 . The K 1 -intermediate at the end of the ultrafast measurement (1.8 ns), remains present at the beginning of the flash photolysis time scale (450 ns), showing that K 1 is populated throughout the ns timescale and no photocycle intermediate is missed due to the experimental time gap (Fig.…”

“…K 1 decays within the early µs-range (lifetime distribution in the range of 5-10 µs) to form the K 2 -intermediate. This intermediate state is considered to be K-like because of the more pronounced red-shift compared to the known L-intermediates of other microbial rhodopsins 30,31 . The transition to the blue-shifted M-intermediate then occurs within ~1 ms.…”

Calcium-Sensitive Microbial Rhodopsin VirChR1: A Femtosecond to Second Photocycle Study

“…Therefore, we concluded that both intermediates are populated simultaneously. Such a feature is common for microbial rhodopsins and was observed various times in the past. − On the late microsecond to early millisecond time scale, another red-shifted photocycle intermediate P 5 is formed, which decays to form the final photocycle intermediate P 6 at ∼10 ms. Ultimately, the protein recovers its parent state after ∼6 s. Intermediates P 5 and P 6 are considered to be especially spectrally similar.…”