Spectral Tuning in Bacteriorhodopsin in the Absence of Counterion and Coplanarization Effects

Abstract

The basis for wavelength regulation in bacteriorhodopsin (BR) and retinylidene proteins in general has been studied for decades but is still only partially understood. Here we report the preparation and spectroscopic characterization of BR analogs aimed at investigating the existence of spectral tuning mechanisms other than the two widely accepted mechanisms, weakened counterion interactions and ring/chain coplanarization. We synthesized two novel retinal analogs containing a saturated 13-14 bond, which interrupts the interaction of the protein counterions with the chromophore conjugation system. Furthermore, one of the analogs has a planar polyene system so that the contribution to the red shift of BR by retinal ring/chain coplanarization is also absent. We incorporated these analogs into bacterioopsin and discovered a sizable amount of red shift, which can be accounted for by interactions between the polar or polarizable groups of the protein and the retinal polyene chain. Our results suggest that the wavelength regulation in BR is achieved by synergistic chromophore/protein interactions including ring/ chain coplanarization, excited state stabilization by polar or polarizable protein side chains located along the polyene chain, and weakened counterion interactions near the Schiff base positive charge. Bacterioopsin (BO)1 tunes the absorption maximum ( max ) of a retinal protonated Schiff base (PSB) from 440 nm in methanol to 568 nm in bacteriorhodopsin (BR). The mechanism of wavelength regulation in BR and in retinylidene proteins in general has been a subject of intensive investigation. Nakanishi et al.(1) introduced the term opsin shift (OS) to refer to the energy difference (in cm Ϫ1

Attractant and Repellent Signaling Conformers of Sensory Rhodopsin−Transducer Complexes

“…Hence, the absorption spectrum of the outwardly connected Schiff base (repellent receptor) conformer is blue-shifted compared to that of the inwardly connected (attractant) conformer. The bluer absorption of the low-p K a conformer with ionized Asp76 is expected from a stronger interaction of Asp76 with the protonated Schiff base (16). …”

“…The absorption spectral maxima of the two conformers differ by 5−10 nm. This difference is eliminated by the Asp76Asn mutation or Asp76 neutralization at low pH and is thereby likely attributable to the difference in counterion charge interaction with the Schiff base proton (16). A remarkable finding in this investigation is that in the absence of ionized Asp76, the wild-type and inverted mutant SRI−HtrI complexes, which have greatly different proportions of attractant and repellent conformers, exhibit indistinguishable absorption spectra (Figure 5).…”

Characterization of an Unconventional Rhodopsin from the Freshwater Actinobacterium Rhodoluna lacicola

“…However, the absorbance maximum of R. lacicola ActR is slightly blueshifted relative to those of the characterized examples from two Octadecabacter species (533 Ϯ 1 nm and 535 Ϯ 1 nm [20]), Salinibacter ruber (560 nm [18]), and Gloeobacter violaceus (540 nm [19]). While the leucine residue is primarily responsible for spectral tuning to green light, the additional variability in observed absorbance maxima of different XR family proteins may be attributed to differences in solution pH (17), which can affect the protonation state of the rhodopsin, and the retinal-binding pocket protein microenvironment (44).…”

Microbial and Animal Rhodopsins: Structures, Functions, and Molecular Mechanisms

“…Other factors defining the spectral tuning of individual rhodopsins are given by chromophore–protein interactions such as electrostatic interactions with charged and polar amino acids, termed electrostatic tuning and extensively studied, first using retinal analogues, 29−32 and, later, site-directed mutagenesis. 33−35 Electrostatic tuning was elegantly demonstrated in a model system based on cellular retinol-binding protein II.…”