Fluorescence Enhancement of a Microbial Rhodopsin via Electronic Reprogramming

Abstract

The engineering of microbial rhodopsins with enhanced fluorescence is of great importance in the expanding field of optogenetics. Here we report the discovery of two mutants (W76S/Y179F and L83Q) of a sensory rhodopsin from the cyanobacterium Anabaena PCC7120 with opposite fluorescence behavior. In fact, while W76S/Y179F displays, with respect to the wild-type protein, a nearly tenfold increase in red-light emission, the second is not emissive. Thus, the W76S/ Y179F, L83Q pair offers an unprecedented opportunity for the investigation of fluorescence enhancement in microbial rhodopsins, which is pursued by combining transient absorption spectroscopy and multi-configurational quantum chemistry. The results of such an investigation point to an isomerization-blocking electronic effect as the direct cause of instantaneous (subpicosecond) fluorescence enhancement.

a-ARM: Automatic Rhodopsin Modeling with Chromophore Cavity Generation, Ionization State Selection, and External Counterion Placement

“…As observed in Figure 5A, the general trend for wild type and Rh mutants models is qualitatively reproduced, mostly displaying blue-shifted absorption similar to the results of the original ARM. 20,52,54,55 Actually, as can be seen in Figure 5B, 30 out of the 39 studied rhodopsins (77%) exhibit blue-shifted errors lower than 3 kcal mol -1 , 6 (15%) higher than 5 kcal mol -1 , and only 3 (8%) present red-shifted values of just few (0.5-1.6) kcal mol -1 . More specifically, among the m-set, BPR and ChR C1C2…”

“…The current version of ARM has been tested for the prediction of trends in λ max a of a limited set of wild type and mutant vertebrate, invertebrate, and microbial rhodopsins, 10,20,52,54,55 showing good agreement with experimental data. The required input includes (A) an X-ray crystallographic structure or comparative model of the protein in PDB (Protein Data Bank) format, 56,57 (B) a list of residues forming the chromophore cavity, (C) the protonation states of ionizable side chains, and (D) the position of extracellular (OS) and intracellular (IS) counterions.…”

“…2 Furthermore, the protein functions are invariably initiated by the photoisomerization of the chromophore triggered by the absorption of light of a specific wavelength. [8][9][10][11][12] The molecularlevel understanding of how variations in the amino acid sequence can modify the functionality of the rhodopsin molecular architecture appears to be not only central to photobiology [13][14][15][16][17][18][19][20] but of importance for the rational design of synthetic mimics [21][22][23] and artificial molecular devices. [24][25][26] In the past, the investigation of how rhodopsin sequence variations modify the residuechromophore interaction, and in turn, the protein light-response has been limited to a relatively, small number of cases.…”

A Unified View on Varied Ultrafast Dynamics of the Primary Process in Microbial Rhodopsins

“…Thee xcited-state PES relevant to the photoisomerization process of various rhodopsins has been extensively discussed theoretically and experimentally. [7,31,36,50,[63][64][65][66][67][68][69] In particular, several recent quantum mechanics/molecular mechanics (QM/MM) calculations provided at heoretical view for the S 1 PES based on the mixing of the S 1 and S 2 states. [31,65,66,69] In this view,t he S 1 state has the polar 1B u electronic character that facilitates ultrafast isomerization due to the energetically down-hill nature along the isomerization coordinate,whereas the higher S 2 state has the nonpolar 2A g electronic character that prohibits the isomerization due to its bound-state nature.…”

“…[25] As more rhodopsins were discovered, aw ider variety of excited-state relaxation dynamics were observed. Fore xample,p roteorhodopsin (PR), [13,[26][27][28][29] Anabaena sensory rhodopsin [16,30,31] and KR2 [18] exhibit complicated excited-state decays that contain several fast (< 1ps) and slow (> 1ps) decay components.Sofar, this wide variety of the multi-phasic excited-state relaxation dynamics observed for different rhodopsins has been mainly explained with the branching of the relaxation pathways on the S 1 Recently,our group studied the primary photoreaction of novel sodium (Na + )-pumping rhodopsin KR2 using femtosecond time-resolved absorption (TA) spectroscopy. [38] It was found that the excited state of KR2 exhibits am ulti-phasic decay that consists of afast (< 1ps) decay and long-lived slow (> 1ps) decays.I nterestingly,t heir relative amplitude drastically changes around the pH corresponding to the pK a of Asp116, the counterion of the PRSB chromophore.T his result indicated that the multi-phasic excited-state decay in KR2 is attributable to the structural heterogeneity in the ground state,that is,the acid-base equilibrium of counterion, Asp116.…”