Effect of point mutations on the ultrafast photo-isomerization of Anabaena sensory rhodopsin

Abstract

Anabaena sensory rhodopsin (ASR) is a particular microbial retinal protein for which light-adaptation leads to the ability to bind both the all-trans, 15-anti (AT) and the 13-cis, 15-syn (13C) isomers of the protonated Schiff base of retinal (PSBR). In the context of obtaining insight into the mechanisms by which retinal proteins catalyse the PSBR photo-isomerization reaction, ASR is a model system allowing to study, within the same protein, the protein-PSBR interactions for two different PSBR conformers at the same time. A detailed analysis of the vibrational spectra of AT and 13C, and their photo-products in wild-type ASR obtained through femtosecond (pump-) four-wave-mixing is reported for the first time, and compared to bacterio- and channelrhodopsin. As part of an extensive study of ASR mutants with blue-shifted absorption spectra, we present here a detailed computational analysis of the origin of the mutation-induced blue-shift of the absorption spectra, and identify electrostatic interactions as dominating steric effects that would entail a red-shift. The excited state lifetimes and isomerization reaction times (IRT) for the three mutants V112N, W76F, and L83Q are studied experimentally by femtosecond broadband transient absorption spectroscopy. Interestingly, in all three mutants, isomerization is accelerated for AT with respect to wild-type ASR, and this the more, the shorter the wavelength of maximum absorption. On the contrary, the 13C photo-reaction is slightly slowed down, leading to an inversion of the ESLs of AT and 13C, with respect to wt-ASR, in the blue-most absorbing mutant L83Q. Possible mechanisms for these mutation effects, and their steric and electrostatic origins are discussed.

Mapping the ultrafast vibrational dynamics of all-transand 13-cisretinal isomerization in Anabaena Sensory Rhodopsin

“…1(a)). The non-oscillatory contributions were subtracted [5] from each DFWM transient before FFT ( Fig. 2(a)-(b)) was performed.…”

“…The DFWM signal, probed at 590 nm, shows the characteristic vibrational frequencies (Fig. 2a) of the GS isomers both in dark adapted (DA) and light adapted (LA) conditions [5] which contain almost pure AT (97%) and major percentage of 13C isomer (64%) respectively. The modes around 1000, 1100-1300 and 1500 cm -1 are usually assigned to CH3 rock, C-C and C=C stretching vibrations.…”

Anabaena Sensory Rhodopsin: Effect of point mutations on PSBR photo-isomerization speed

“…However, the absolute QY values are the lowest ever reported for retinal proteins (< 25%) demanding an extended study on point mutated proteins for a deeper understanding. We report on point-mutated proteins showing an up to ~2 fold reduction of the AT isomer ESL with respect to WT-ASR, with a trend indicating a correlation between ESL and absorption maximum wavelength (fig 1a) [4]. In contrary, the 13C isomer ESL appears less sensible to mutations leading in some cases to even longer ESL than in WT-ASR.…”

“…TA experiments probing the UV/VIS part of the spectrum highlighting the prominent excited-state or photoproduct absorptions (ESA and PA) were reported for these mutants [4]. We concentrate here on the near-IR stimulated emission (SE), which allows to retrieve the ESL's without any interfering ground state signal (fig 1b).…”

Fluorescence Enhancement of a Microbial Rhodopsin via Electronic Reprogramming

“…In all cases, we assume that during such a short time, the trajectories describe the average evolution of population on the lowest excited states (S 1 and S 2 ). 30 As shown in Fig. 4A, L83Q reaches the photochemically relevant S 1 /S 0 CI and decays to S 0 in ca.…”