Abstract
Microbial rhodopsins have become an important tool in the field of optogenetics. However, effective in vivo optogenetics is in many cases severely limited due to the strong absorption and scattering of visible light by biological tissues. Recently, a combination of opsin site-directed mutagenesis and analog retinal substitution has produced variants of proteorhodopsin which absorb maximally in the near-infrared (NIR). In this study, UV-Visible-NIR absorption and resonance Raman spectroscopy were used to study the double mutant, D212N/F234S, of green absorbing proteorhodopsin (GPR) regenerated with MMAR, a retinal analog containing a methylamino modified β-ionone ring. Four distinct subcomponent absorption bands with peak maxima near 560, 620, 710 and 780 nm are detected with the NIR bands dominant at pH <7.3, and the visible bands dominant at pH 9.5. FT-Raman using 1064-nm excitation reveal two strong ethylenic bands at 1482 and 1498 cm-1 corresponding to the NIR subcomponent absorption bands based on an extended linear correlation between λmax and γC = C. This spectrum exhibits two intense bands in the fingerprint and HOOP mode regions that are highly characteristic of the O640 photointermediate from the light-adapted bacteriorhodopsin photocycle. In contrast, 532-nm excitation enhances the 560-nm component, which exhibits bands very similar to light-adapted bacteriorhodopsin and/or the acid-purple form of bacteriorhodopsin. Native GPR and its mutant D97N when regenerated with MMAR also exhibit similar absorption and Raman bands but with weaker contributions from the NIR absorbing components. Based on these results it is proposed that the NIR absorption in GPR-D212N/F234S with MMAR arises from an O-like chromophore, where the Schiff base counterion D97 is protonated and the MMAR adopts an all-trans configuration with a non-planar geometry due to twists in the conjugated polyene segment. This configuration is characterized by extensive charge delocalization, most likely involving nitrogens atoms in the MMAR chromophore.
Highlights
Microbial rhodopsins are retinal containing, seven-helix transmembrane proteins that absorb UV and visible light
The absorption spectrum in the 250–950 nm region of the mutant green absorbing proteorhodopsin (GPR)-D212N/F234S regenerated with MMAR (GPR-DNFS:MR) reconstituted into E. coli polar lipids (ECPL) lipid membrane at pH 5, 7 and 9.5 and deposited as a hydrated multilamellar film onto a BaF2 window which is part of a sealed cell is shown in Fig 2
A weak band near 415 nm appears for all GPRs regenerated with MMAR which is likely to arise from residual cytochrome impurities that are often difficult to remove during E. coli membrane protein purification using Ni-NTA agarose His-tag affinity chromatography [25, 55]
Summary
Microbial rhodopsins (classified as Type 1 rhodopsins) are retinal containing, seven-helix transmembrane proteins that absorb UV and visible light. The visible absorption maxima of PRs are clustered near 520 nm (green proteorhodopsin; GPR) or 490 nm (blue proteorhodopsin; BPR) [18, 20, 21]. They all share with BR several key conserved amino acid residues including Asp in helix C (Asp in BR), which functions as the Schiff base (SB) counterion and proton acceptor, Glu108 (Asp 96 in BR), the Schiff Base (SB) proton donor and Lys231 in helix G (Lys216 in BR) which forms a SB with the retinylidene chromophore (Fig 1). Other residues such as His (helix B) have no counterpart in BR and may serve as part of a proton relay mechanism through direct interaction with Asp97 [22,23,24], as well as in inter-subunit interaction in oligomer formation [23]
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