Raman spectroscopy of a near infrared absorbing proteorhodopsin: Similarities to the bacteriorhodopsin O photointermediate

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.

Optical Switching Between Long‐lived States of Opsin Transmembrane Voltage Sensors

“…In support of this band assignment, a strong 1567 cm −1 band appears in the resonance Raman spectrum of the BR M intermediate ( 48 , 49 ). The lower frequency of the NovArch M‐like state compared to BR‐M (1559 vs 1567 cm −1 ) is consistent with the empirical linear correlation between wavelength of absorption ( λ max ) and ethylenic frequency (ν C=C ) as described previously ( 34 , 50 , 51 , 52 , 53 , 54 ) (Figure S8 ). Notably, a band at 1574 cm −1 assigned to the C = C stretching frequency of the UV absorbing state (380 nm) of HKR1 also fits this linear correlation ( 55 ).…”

Surface-enhanced Raman spectroscopy for bioanalysis and diagnosis

“…The exci-tation wavelength used also "selects" only the particular species by resonance conditions. For instance, resonant Raman or SERS spectroscopy was also used for studying many "intrinsically colored" biomolecules such as chlorophylls, 35 haem-containing proteins, 36 carotenoids, 37 rhodopsin, 38 and flavin nucleotides, 39 and with the aid of robust chemometric tools, their accurate detection and discrimination in living cells was successfully reported. 40 There are numerous opportunities to use SERS in real multiplex detection for screening biological processes, real-time mapping of living cells, etc.…”

Collective exchange processes reveal an active site proton cage in bacteriorhodopsin

“…2a, d) of BR wildtype purple membranes. We further investigated in this way BR mutated at the proton donor of the RSB (D96), the acceptor of the RSB proton (D85) and the proton shuttle R82: D96N, in which the reprotonation of the RSB is dramatically slowed down 32 , D85T that features a red-shift of the BR-absorption and complete inactivation of proton pumping 33,34 , and R82Q, in which BR-absorption is red-shifted, the RSB-pK a lowered, and proton shuttling from D85 to the proton release group severely hampered 35,36 .…”