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

Article https://doi.org/10.1038/s41467-022-33953-y Retinal chromophore charge delocalization and confinement explain the extreme photophysics of Neorhodopsin Received: 21 April 2022 Check for updates 1234567890():,; 1234567890():,; Accepted: 7 October 2022 Riccardo Palombo1,2, Leonardo Barneschi1, Laura Pedraza-González1, Daniele Padula 1, Igor Schapiro 3 & Massimo Olivucci 1,2 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. Modern neuroscience requires membrane-localized signaling tools1,2 that could emit intense fluorescence upon irradiation with red light. However, until recently, the available tools, based on engineered microbial rhodopsins, could only generate weak fluorescence signals that impair their performance. At the molecular level, the optical properties of microbial rhodopsins owe to the presence of a covalently bounded all-trans retinal protonated Schiff base (rPSB) chromophore and its interaction with the surrounding protein environment. Therefore, a deep molecular comprehension of the factors dictating such properties is highly desirable. In this regard, few studies3–5 have formulated rules for tailoring the absorption and emission properties of the retinal chromophore based on the effects of homogeneous electrostatic fields acting on isolated chromophores or via chromophore chemical modifications. However, it is expected that a simple electrostatic picture could not be sufficient to explain the origin of these properties in the complex environment offered by the protein cavity since other factors like non-homogeneous electrostatic fields or chromophore-cavity steric effects could play an important role. In 2020 the discovery of Neorhodopsin (NeoR) offered an unprecedent case study that could potentially expand our comprehension of red-shifted and highly fluorescent rhodopsins. NeoR is a rhodopsin guanylyn-cyclase (RGC) expressed in the Rhizoclosmatium globosum from Chytridiomycota, the only phylium of fungi producing motile and flagellated spores (zoospores)6,7. It heterodimerizes with other two RGCs, called RGC1 and RGC2, that have sensitivity in the blue-green spectrum with 550 and 480 nm absorption maxima (λamax), respectively. In contrast, NeoR displays the strongest bathocromic shift among all known microbial rhodopsins, yielding an extremely red-shited (λamax = 690 nm) absorption band. Such a band is mirrored by an intense emission band with a maximum (λfmax) at 707 nm yielding Stokes shift of only 17 nm (350 cm−1). The emission brightness is quantified by a fluorescence quantum yield (FQY) of 20% and by an extinction coefficient (ϵ) of 129,000 M−1 cm−1. In addition, the excited state lifetime (ESL) of 1.1 ns points to a slow excited state deactivation. The FQY of NeoR, only ca. four times weaker than that of the green fluorescent protein8 (GFP), represents an anomaly in the rhodopsin superfamily and suggests an evolution-driven origin. More specifically, 1 Dipartimento di Biotecnologie, Chimica e Farmacia, Università di Siena, via A. Moro 2, I-53100 Siena, Italy. 2Department of Chemistry, Bowling Green State University, Bowling Green, OH 43403, USA. 3Fritz Haber Center for Molecular Dynamics, Institute of Chemistry, The Hebrew University of Jerusalem, 9190401 e-mail: Jerusalem, Israel. Nature Communications | (2022)13:6652 1 Article https://doi.org/10.1038/s41467-022-33953-y 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. BLAPSB is the -C14-C15 and C15-N bond lengths difference. since the emission competes with the photoisomerization of its rPSB chromophore, a presently unknown adaptation process must have decreased the efficiency of the protein function. This hypothesis is in line with the fact that wild-type (WT) rhodopsins commonly exhibit FQYs spanning the 0.0001%–0.01%9–12 range while engineering efforts yielded variants with only modest increases up to a 1.2% value13–16. Deciphering how natural evolution in NeoR has tuned these extreme spectroscopic properties of the rPSB chromophore could expand our ability to design optogenetic tools with augmented functionality. Therefore, the modeling of NeoR represents a new promising learning opportunity that can be also used to assess the transferability of the rules mentioned above. In particular, NeoR offers the opportunity to disclose the molecular-level mechanism controlling the branching between fluorescence emission and photoisomerization. Such branching, which is schematically illustrated in Fig. 1a for all-trans rPSB, has been shown to dominate the fluorescence modulation in a set of GFP-like protein variants8,17. More specifically, in these systems, the FQY appears to be directly proportional to the energy barrier (EfS1) controlling both access to a conical intersection (CoIn) located along the first singlet excited state (S1) isomerization coordinate and the decay to the ground state (S0). Here we assume that the same mechanism operates in NeoR is then used as a “laboratory” model for proposing a mechanism capable to connect sequence variation and rPSB emission. To do so, we also assume, in line with the evidence coming from a set of Arch3 variants displaying enhanced fluorescence18,19, that the NeoR emission is a one-photon process and that, therefore, originates directly from its dark adapted state (DA). In order to pursue the object (...truncated)