Abstract
Heliorhodopsins are a recently discovered diverse retinal protein family with an inverted topology of the opsin where the retinal protonated Schiff base proton is facing the cell cytoplasmic side in contrast to type 1 rhodopsins. To explore whether light-induced retinal double-bond isomerization is a prerequisite for triggering protein conformational alterations, we utilized the retinal oxime formation reaction and thermal denaturation of a native heliorhodopsin of Thermoplasmatales archaeon SG8-52-1 (TaHeR) as well as a trans-locked retinal analogue (TaHeRL) in which the critical C13=C14 double-bond isomerization is prevented. We found that both reactions are light-accelerated not only in the native but also in the “locked” pigment despite lacking any isomerization. It is suggested that light-induced charge redistribution in the retinal excited state polarizes the protein and triggers protein conformational perturbations that thermally decay in microseconds. The extracted activation energy and the frequency factor for both the reactions reveal that the light enhancement of TaHeR differs distinctly from the earlier studied type 1 microbial rhodopsins.
Highlights
Microbial and animal rhodopsin protein families are both composed of seven transmembrane α-helices with the C-terminus facing inside of the cell.[1]
The proposal was based on studies with bacteriorhodopsin using atomic force sensing (AFS) methodology and electron paramagnetic resonance (EPR) spectroscopy of apo-bacteriorhodopsin membrane hosting synthetic nonisomerizable chromophores.[5−7] Light acceleration of the EPR-probe redox reaction indicated that the protein experiences structural changes even without retinal double-bond isomerization.[5]
The overall sequence identity of the HeR derived from Thermoplasmatales archaeon SG8-52-1 (TaHeR), which is employed in the current study, is only ∼8% of the type 1 microbial rhodopsin family.[13]
Summary
Microbial and animal rhodopsin protein (type 1 and type 2, respectively) families are both composed of seven transmembrane α-helices with the C-terminus facing inside of the cell.[1]. The retinal chromophore undergoes light-induced double-bond isomerization from an all-trans to 13-cis isomer followed by a sequence of protein conformational alterations.[1−4]. The overall sequence identity of the HeR derived from Thermoplasmatales archaeon SG8-52-1 (TaHeR), which is employed in the current study, is only ∼8% of the type 1 microbial rhodopsin family.[13] Considering its long-lifetime photocycle, it has been suggested that HeR acts as a signaling photoreceptor, though its exact function is not clear yet.[12] HeR possesses a unique topology in which the protein N-termini faces the cell cytoplasm, an inverted orientation relative to both type 1 and type 2 rhodopsins This unique topology and a very dissimilar residue sequence raise the question whether light-induced protein conformational alterations can occur in TaHeR without the retinal double-bond isomerization. To determine the activation energy (Ea) and the frequency factor (A) of the processes, the obtained reaction rates in the dark and in light at various temperatures (T) were fitted to the Arrhenius equation
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