Abstract
Bistable opsins are photopigments expressed in both invertebrates and vertebrates. These light-sensitive G-protein-coupled receptors undergo a reversible reaction upon illumination. A first photon initiates the cis to trans isomerization of the retinal chromophore—attached to the protein through a protonated Schiff base—and a series of transition states that eventually results in the formation of the thermally stable and active Meta state. Excitation by a second photon reverts this process to recover the original ground state. On the other hand, monostable opsins (e.g., bovine rhodopsin) lose their chromophore during the decay of the Meta II state (i.e., they bleach). Spectroscopic studies on the molecular details of the two-photon cycle in bistable opsins are limited. Here, we describe the successful expression and purification of recombinant rhodopsin-1 from the jumping spider Hasarius adansoni (JSR1). In its natural configuration, spectroscopic characterization of JSR1 is hampered by the similar absorption spectra in the visible spectrum of the inactive and active states. We solved this issue by separating their absorption spectra by replacing the endogenous 11-cis retinal chromophore with the blue-shifted 9-cis JSiR1. With this system, we used time-resolved ultraviolet-visible spectroscopy after pulsed laser excitation to obtain kinetic details of the rise and decay of the photocycle intermediates. We also used resonance Raman spectroscopy to elucidate structural changes of the retinal chromophore upon illumination. Our data clearly indicate that the protonated Schiff base is stable throughout the entire photoreaction. We additionally show that the accompanying conformational changes in the protein are different from those of monostable rhodopsin, as recorded by light-induced FTIR difference spectroscopy. Thus, we envisage JSR1 as becoming a model system for future studies on the reaction mechanisms of bistable opsins, e.g., by time-resolved x-ray crystallography.
Highlights
Animal opsins are light-sensitive G-protein-coupled receptors (GPCRs) mainly involved in vision and circadian clock entrainment [1]. In these GPCRs, photon absorption results in the isomerization of a retinal chromophore covalently linked to the protein through a protonated Schiff base
Schiff base during Meta II decay leads to the eventual loss of the retinal chromophore
In bistable opsins—such as jumping spider rhodopsin-1 (JSR1) [3]—retinal remains in the protein binding pocket throughout the entire photoreaction [4], and its isomerization leads to the formation of a thermally stable active state [4,5,6,7], suggesting the presence of a protonated Schiff base [4,6]
Summary
Animal opsins are light-sensitive G-protein-coupled receptors (GPCRs) mainly involved in vision and circadian clock entrainment [1]. In bistable opsins—such as jumping spider rhodopsin-1 (JSR1) [3]—retinal remains in the protein binding pocket throughout the entire photoreaction [4], and its isomerization leads to the formation of a thermally stable active state (acid-Meta) [4,5,6,7], suggesting the presence of a protonated Schiff base [4,6]. Illumination of this state recovers the original inactive ground state (Rho). We expect that these results will assist in establishing JSR1 as a model system for the study of novel light-controlled molecular switches in optogenetic applications
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