Factors affecting the absorption maxima of acidic forms of bacteriorhodopsin. A study with artificial pigments

Abstract

The absorption maximum (568 nm) of light-adapted bacteriorhodopsin bR568 undergoes reversible changes after acidification. At pH 2.9, the absorption shifts to 605 nm (forming bR605) and it blue shifts to 565 nm, after further acidification to pH approximately 0.5 (forming bR565). Molecular models accounting for such acid-induced changes are relevant to the structure and function of bacteriorhodopsin. In the present study we approached the problem by applying artificial bR pigments based on selectively modified synthetic retinals. This may allow direct identification of the specific regions in the retinal binding site where the above changes in the protein-retinal interactions take place. We investigated the spectroscopic effects of acid in a variety of artificial pigments, including cyaninelike retinals, retinals bearing bulky groups at C4, short polyenes, and retinals in which the beta-ionone ring was substituted by aromatic rings. The results provide direct evidence for the hypothesis that the generation of bR605 is due to changes in polyene-opsin interactions in the vicinity of the Schiff base linkage. The second transition (to bR565) was not observed in artificial pigments bearing major changes in the ring structure of the retinal. Two approaches accounting for this observation are presented. One argues that the generation of bR565 is associated with acid-induced changes in retinal-protein interactions in the vicinity of the retinal ring. The second involves changes in polyene-opsin interactions in the vicinity of the Schiff base linkage. For both bRw and bRN5, our results do not discriminate between the direct titration of negative or dipolar protein groups in the binding site and changes in the retinal-protein interactions induced by changes in the protein structure outside of the binding site.

Synthetic retinal analogues modify the spectral and kinetic characteristics of microbial rhodopsin optogenetic tools

“…Reconstitution of Arch3 and four ChR variants with A2 retinal, which carries an extra double bond, resulted in functional pigments with enhanced longwavelength sensitivity 29 . Extensive work on bacteriorhodopsin (BR) [45][46][47][48][49] using synthetic retinal analogues suggests that many other retinal analogues, particularly those with ring modifications, might be applicable for the purpose of chromophore substitution. However, in most cases, it was only shown that the absorption maximum was shifted, without analysing the rhodopsin function.…”

Electronic State Mixing Controls the Photoreactivity of a Rhodopsin with all-trans Chromophore Analogues

“…In the past, bacteriorhodopsin (bR) hosting different artificial rPSB pigments have been employed in various experimental studies. 9–13 One of such studies have provided evidence that electron withdrawing groups such as CF 3 inhibit the photoisomerization. 10 However, a computational/mechanistic study looking at which specific effects (i.e.…”

Directed Evolution of a Bright Near-Infrared Fluorescent Rhodopsin Using a Synthetic Chromophore

“…Chromophore-dependent microbial opsins provide an excellent platform for this approach since modified retinals are well accepted and their incorporation can dramatically alter and enhance opsin properties (Albeck et al, 1989; Asato et al, 1990; AzimiHashemi et al, 2014; Gaertner et al, 1981; Ganapathy et al, 2015; Hoischen et al, 1997; Nielsen, 2009; Sineshchekov et al, 2012). We have used a synthetic chromophore, merocyanine retinal, to generate a near-infrared fluorescent protein that is 200 nm red-shifted and at least 16-fold brighter than WT Arch bound to its natural chromophore, ATR.…”

“…With appropriate screens, one could use directed evolution to enhance other properties of interest such as enhanced expression in specific cell types, high voltage sensitivity, or pumping activity using tailored synthetic retinals that are sensitive to specific wavelengths. We anticipate that this general approach will continue to provide fine-tuned properties and functions useful for optogenetic sensors or actuators (AzimiHashemi et al, 2014; Sineshchekov et al, 2012), energy harvesting (Ganapathy et al, 2015; Walter et al, 2010), or cell labeling and imaging applications (Albeck et al, 1989; Hoischen et al, 1997). …”

“…Spectral properties of the natural ATR chromophore (Figure 1 A , Compound 1) can be modulated by adding electron-withdrawing groups (Gaertner et al, 1981; Hendrickx et al, 1995) or changing the length of the conjugated π-bond system (Albeck et al, 1989; Nielsen, 2009). In particular, retinal analogs with extended conjugation have been shown to red-shift the absorption maxima of rhodopsins up to hundreds of nanometers, well into the NIR window (Asato et al, 1990; Sineshchekov et al, 2012).…”