Red-Tuning of the Channelrhodopsin Spectrum Using Long Conjugated Retinal Analogues

Abstract

As optogenetic studies become more popular, the demand for red-shifted channelrhodopsin is increasing, because blue-green light is highly scattered or absorbed by animal tissues. In this study, we developed a red-shifted channelrhodopsin by elongating the conjugated double-bond system of the native chromophore, all -trans-retinal (ATR1). Analogues of ATR1 and ATR2 (3,4-didehydro-retinal) in which an extra C═C bond is inserted at different positions (C6-C7, C10-C11, and C14-C15) were synthesized and introduced into a widely used channelrhodopsin variant, C1C2 (a chimeric protein of channelrhodopsin-1 and channelrhodopsin-2 from Chlamydomonas reinhardtii). C1C2 bearing these retinal analogues as chromophores showed broadened absorption spectra toward the long-wavelength side and photocycle intermediates similar to the conducting state of channelrhodopsin. However, the position of methyl groups on the retinal polyene chain influenced the yield of the pigment, absorption maximum, and photocycle pattern to a variable degree. The lack of a methyl group at position C9 of the analogues considerably decreased the yield of the pigment, whereas a methyl group at position C15 exhibited a large red-shift in the absorption spectra of the C1C2 analogue. Expansion of the chromophore binding pocket by mutation of aromatic residue Phe265 to Ala improved the yield of the pigment bearing elongated ATR1 analogues without a great alteration of the photocycle kinetics of C1C2. Our results show that elongation of the conjugated double-bond system of retinal is a promising strategy for improving the ability of channelrhodopsin to absorb long-wavelength light passing through the biological optical window.

Rapid Oxidation Following Photoreduction in the Avian Cryptochrome4 Photocycle

“…We performed singular value decomposition (SVD) of spectroscopic data obtained in the presence of DTT (Figure 1A), as previously reported. 56 Ideally, the spectral changes of reactions with different time constants can be decomposed into B spectra in the SVD analysis (Figure S5). B spectra are the decay-associated difference spectra.…”

“…Changes to the transient absorption of samples were observed using a laboratory-built, time-resolved spectrophotometry system, the same as previously used. 56 For photoexcitation, a xenon lamp (Short Arc Power Flash model-SA-200; Nissin Electronic, Kyoto, Japan) with a blue (Figure S2A) and a yellow (Figure S2B) filter were used. Samples were kept at 20 °C during measurement.…”

Emerging Diversity of Channelrhodopsins and Their Structure-Function Relationships

“…Extension of the chromophore π-system can alternatively be achieved by elongation of the polyene chain upon the insertion of a vinylene group. A series of such analogs has been tested in purified CCRs mutated to improve their affinity for such compounds (Shen et al, 2018 ; Okitsu et al, 2020 ). Channel activity of these molecules has not yet been tested, but measurements of flash-induced absorbance changes have revealed that the kinetics of the photocycle was altered, as compared to that of all- trans retinal-bound proteins.…”

Synthesis of One Double Bond-Inserted Retinal Analogs and Their Binding Experiments with Opsins: Preparation of Novel Red-Shifted Channelrhodopsin Variants

“…1). All analogs except for 3a were incorporated into C1C2, which is one of the most widely used ChR variants in optogenetics (an absorption maximum at 477 nm), 17) and among the newly created ChRs, A2-10ex-C1C2 showed the longest absorption maximum at 510 nm. 18) These results indicated that polyene chain-elongated retinals could be incorporated into the retinal-binding pocket of C1C2.…”