The Two-Photon Reversible Reaction of the Bistable Jumping Spider Rhodopsin-1

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.

Crystal structure of jumping spider rhodopsin-1 as a light sensitive GPCR

“…Monostable rhodopsins, e.g., bovine rhodopsin, differ from bistable rhodopsins, e.g., squid and jumping spider rhodopsins, as well as the vertebrate melanopsin involved in the circadian clock regulation, by having a unidirectional isomerization, followed by deprotonation and hydrolysis of the Schiff base. Conversely, in bistable rhodopsins a second photon reisomerizes the retinal while the Schiff base remains protonated throughout the photocycle (8).…”

“…Illumination eventually bleaches monostable rhodopsins by deprotonation and hydrolysis of the Schiff base and subsequent release of the chromophore (6). Bistable rhodopsins, on the other hand, undergo a 2-photon bidirectional photoreaction ( cis-trans/trans-cis ) in which the Schiff base remains protonated and the retinal bound throughout the photocycle (4, 7, 8). Over the years, biophysical and structural characterization of bovine rhodopsin (monostable) and squid rhodopsin (bistable) have provided insights into these mechanistic differences.…”

Immobilization of arrestin-3 on different biosensor platforms for evaluating GPCR binding

“…Wild-type JSR1 with a C-terminal 1D4 epitope tag was expressed and purified as described in Ehrenberg et al 27 Briefly, a suspension culture of HEK293S GnTI-cell line stably expressing JSR1 was cultivated in FreeStyle 293 expression medium to 2 Â 10 6 cells per mL and then harvested. Frozen cells were mechanically homogenized in 50 mM HEPES, 140 mM NaCl, and 3 mM MgCl 2 with protease inhibitors (Roche complete EDTA-free Protease Inhibitor Cocktail; Sigma-Aldrich, Buchs, Switzerland) and incubated overnight with 30 mM 9-cis retinal (98% grade, Sigma-Aldrich) overnight at 4 1C in the dark for reconstitution with the chromophore.…”

“…As a model GPCR we selected Jumping Spider Rhodopsin-1 (JSR1), a light-sensitive bistable class A GPCR, which has recently been shown to be highly thermostable in its wild-type form in vitro 26 and is an interesting target for the development of optogenetic tools. 26,27 In fact, as other rhodopsins, JSR1 is easily activated by illumination without the addition of ligands. Moreover, invertebrate rhodopsins are known to be able to bind arrestin in a phosphorylation-independent way upon illumination.…”

“…similarities between JSR1 and other non-light sensitive class A GPCRs 26 suggesting JSR1 as a valid model for in vitro binding studies. 26,27 At the same time, we establish here a platform, which allows an in-depth biophysical characterization of arrestin binding of any GPCR with pharmaceutical relevance.…”