Retinal chromophore charge delocalization and confinement explain the extreme photophysics of Neorhodopsin

Abstract

The understanding of how the rhodopsin sequence can be modified to exactly modulate the spectroscopic properties of its retinal chromophore, is a prerequisite for the rational design of more effective optogenetic tools. One key problem is that of establishing the rules to be satisfied for achieving highly fluorescent rhodopsins with a near infrared absorption. In the present paper we use multi-configurational quantum chemistry to construct a computer model of a recently discovered natural rhodopsin, Neorhodopsin, displaying exactly such properties. We show that the model, that successfully replicates the relevant experimental observables, unveils a geometrical and electronic structure of the chromophore featuring a highly diffuse charge distribution along its conjugated chain. The same model reveals that a charge confinement process occurring along the chromophore excited state isomerization coordinate, is the primary cause of the observed fluorescence enhancement.

Fig. 1. Geometrical and electronic structure changes in NeoR.

a Schematic representation of the hypothetic S₀ and S₁ energy changes occurring along the S₁ relaxation that involves the bond length alternation (BLA, quantified by the difference between the average of the double-bond lengths and the average of the single-bond lengths of the conjugated chain) and isomerization (α) coordinates. The rPSB resonance hybrids show a delocalized positive charge at the S₀ and S₁ energy minima corresponding to the Dark Adapted State (DA) and Fluorescent State (FS), respectively. The symbol “δ+“ gives a qualitative measure of the amount of positive charge located along the rPSB-conjugated chain. b Representation of the bond length alternation (BLA) mode and the torsion mode (α) along the C13=C14 double bond. BLA_PSB is the -C14-C15 and C15-N bond lengths difference.

Fig. 2. Choice of the NeoR chromophore counterion and model assessment.

a Overview of the structure of the all-trans rPSB chromophore (orange) and its four potential residue counterions (in green). The lysine residue (in green) bounded to the rPSB chromophore is also displayed. b Computed (CASPT2 level) maximum absorption wavelength (λ^a_max), maximum emission wavelength (λ^f_max) and relaxation energy (E^r) of NeoR with varying counterion choices. c Correlation between experimental (Obs. ∆E_S0–S1) and computed (Comp. ∆E_S0–S1) values of vertical excitation energies defining λ^a_max in the wild type (indicated as WT) and a set of NeoR mutants. d Superimposition of experimental and computed (dotted line) absorption band of wild type NeoR. The experimental band has been digitalized from the corresponding ref. .

Fig. 3. Electronic and geometrical character of S₁ relaxation in NeoR chromophore.

a Representation of the two limiting resonance formulas adopted to describe the electronic character of the rPSB chromophore. b Electron density variation (δρ_abs) characterizing the vertical S₀→S₁ transition from the Dark Adapted State (DA). Blue and red clouds correspond to electron density decrease and increase respectively. Isovalue set to 0.002 a.u. The associated resonance formulas correspond to resonance hybrids also anticipated in Fig. 1a. Blue bubbles represent the QM positive charge (in e unites). Only absolute values > 0.05 e are reported. As indicated by the red box, the total charge residing in the -C14-C15-N-Cε- rPSB fragment is also given. c Same data for the S₁→S₀ emission from the Fluorescent State (FS). d Geometrical comparison between DA and FS rPSB structures. The arrows indicate the dominant geometrical change corresponding, clearly, to a variation in the bond length alternation (BLA, see definition in the caption of Fig. 1) in a region of the conjugated chain distant from the Schiff base moiety. The relevant bond lengths are given in Å.

Fig. 4. S₁ energy, charge and BLA profiles along the photoisomerization of, from left to right, C13=C14, C11=C12, C9=C10 and C7=C8 double bonds.

a CASPT2 S₁ energy profiles computed in presence of the full protein environment (orange squares), after setting to zero the MM charges of the entire protein (gold squares), after setting to zero only the MM charges of the rPSB counterion (i.e., E141, gray squares) and after removing the whole protein in a full QM calculation (i.e., in vacuo, empty squares). b Evolution of S₀ and S₁ Charge_PSB (the charge residing in the C14-C15-N-Cε rPSB moiety) and evolution of BLA_PSB (C14-C15 and C15-N bond lengths difference, see Fig. 1b).

Fig. 5. Origin of the E^f_S1 barrier in NeoR.

a Representation of the increase in the distance between the negative and positive charge centroids due to the positive charge confinement along the C13=C14 and C9=C10 photoisomerization paths. Comparison between electrostatic potential (ESP) maps indicates that along the C9=C10 coordinate the extent of the confinement is less pronounced being the charge at the CoIn spread on a longer rPSB chromophore moiety (i.e., C10-C11-C12-C13-C14-C15-N). b Proposed origin of the isomerization barrier in terms of COV energy (H_COV, dashed line) destabilization due to the charge confinement resulting from the mixed [CT] - c’[COV] to the pure [COV] electronic structure change along the S₁ adiabatic energy profile (in orange). We hypothesize that the diabatic energy curves cross halfway along the isomerization coordinate α, which therefore corresponds to the point with the highest diabatic coupling. The such diabatic coupling will then vanish at the CoIn. The left and right panels display the shape of the S₁ and S₀ adiabatic potential energy curves along the BLA_PSB coordinate (see definition in the caption of Fig. 4) at FS (left) and CoIn (right) and are in line with the presented FC→FS and CoIn computations. The BLA_PSB and α coordinates are substantially orthogonal.