NeoR, a near-infrared absorbing rhodopsin

Abstract

The Rhizoclosmatium globosum genome encodes three rhodopsin-guanylyl cyclases (RGCs), which are predicted to facilitate visual orientation of the fungal zoospores. Here, we show that RGC1 and RGC2 function as light-activated cyclases only upon heterodimerization with RGC3 (NeoR). RGC1/2 utilize conventional green or blue-light-sensitive rhodopsins (λ_max = 550 and 480 nm, respectively), with short-lived signaling states, responsible for light-activation of the enzyme. The bistable NeoR is photoswitchable between a near-infrared-sensitive (NIR, λ_max = 690 nm) highly fluorescent state (Q_F = 0.2) and a UV-sensitive non-fluorescent state, thereby modulating the activity by NIR pre-illumination. No other rhodopsin has been reported so far to be functional as a heterooligomer, or as having such a long wavelength absorption or high fluorescence yield. Site-specific mutagenesis and hybrid quantum mechanics/molecular mechanics simulations support the idea that the unusual photochemical properties result from the rigidity of the retinal chromophore and a unique counterion triad composed of two glutamic and one aspartic acids. These findings substantially expand our understanding of the natural potential and limitations of spectral tuning in rhodopsin photoreceptors.

Fig. 1. Phylogeny, model, and activity of the Rhizoclosmatium globosum rhodopsin-guanylyl cyclases (RGCs).

a Unrooted phylogenetic tree of microbial rhodopsins, with red circles indicating bootstrap values >90%; the scale bar represents average amino acid substitutions per site. b Model of R. globosum heterodimeric RGCs: rhodopsins, magenta (RGC1/2) and cyan (NeoR); cyclase, blue; and linker, yellow. c Representative currents from Xenopus oocytes expressing RGC1/2 and NeoR individually or in combination, in response to a 2 s light pulse (yellow box); initial photocurrent slopes are plotted as mean ± standard error (s.e.m.); N = number of biological replicates. d Photocurrent action spectrum (red squares; means ±  standard deviation (s.d.), derived from three biological replicates) of RGC2/NeoR in ND7/23 cells, normalized to the maximum current underlayed by the RGC2 absorption. e Photocurrents (means ± s.e.m., with N = number of biological replicates) of RGC2/NeoR heterodimers generated by 10 ms blue-light (490 nm) flashes of different intensities (as visualized in the inset) in the absence (blue cycles) or presence (red cycles) of 660 nm background light (continuous wave illumination (cwi)).

Fig. 2. Spectral properties of purified rhodopsin domains from the R. globosum RGCs.

a Normalized absorption spectra of the purified recombinant rhodopsin domains from RGC1, RGC2, and NeoR (red and UV-form); picture of the purified NeoR sample, inset. b Reversible bleaching of NeoR₆₉₀ with 680 nm light. c Reversible bleaching of NeoR₃₆₇ with 375 nm light. d Kinetic traces of NeoR bleaching with 680 nm light and subsequent recovery by 375 nm illumination. e Absorption spectra of purified NeoR expressed in media supplemented with A1-retinal or 3,4-dihydro-retinal (A2), as compared to those from biliverdin (BV)-containing iRFP. f Fluorescence spectra of NeoR₆₉₀: excitation (dots) overlaid with the absorbance (red) and emission (green). g Evolution-associated difference spectra (EADS) of fs–ns transient absorption spectra derived from pump-probe experiments, time trace at 708 nm, inset. h Representative confocal images of NeoR fluorescence (red) and the C-terminal eGFP tag (green) in ND7/23 cells. NeoR is reversibly bleached by illumination with 640- and 405 nm light. The reversible bleaching of NeoR fluorescence in single cells was repeated in three independent experiments using five fields of view each. Scale bar, 100 µm.

Fig. 3. Molecular model and spectral features of NeoR mutants.

a Homology model of the NeoR retinal-binding pocket, with the amino acids targeted by mutagenesis drawn as sticks. b Absorption spectra of NeoR mutants; spectra are normalized to the respective UV-form. c Spectral features of NeoR mutants; sw, bimodal switchable; dp, depronotated chromophore; Ф_F, fluorescence quantum yield (in %). d Emission spectra (at 650 nm), and e excitation spectra (at 720 nm) of wild-type (WT) NeoR and NeoR mutants.

Fig. 4. QM/MM Modeling of NeoR.

a Overview of QM/MM excitation energy calculations of eight systems (#1–8) with varying protonation of E136, D140 and E262 as indicated. The total charge of the three carboxylates in each system is given. λ_MAX refers to the absorption maxima as derived from excited-state energy calculation. b Active site from QM/MM-optimized models, with hydrogen bonds represented as dashed lines. c Water intrusion into NeoR as observed during subsequent 300 ns MD simulations with water densities visualized as red meshes. The retinal and amino acid sidechains are taken from the initial structure of the MD simulations.