Chimeric Proton-Pumping Rhodopsins Containing the Cytoplasmic Loop of Bovine Rhodopsin

Kengo Sasaki,Kazuho Yoshida,Keiichi Inoue,Takahiro Yamashita,Yoshinori Shichida,Hideki Kandori,Karl-Wilhelm Koch

doi:10.1371/journal.pone.0091323

Kengo Sasaki, Kazuho Yoshida + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0091323

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Journal: PLoS ONE	Publication Date: Mar 12, 2014
Citations: 16	License type: CC BY 4.0

Affiliation: Nagoya Institute of Technology, Kyoto University

Abstract

G-protein-coupled receptors (GPCRs) transmit stimuli to intracellular signaling systems. Rhodopsin (Rh), which is a prototypical GPCR, possesses an 11-cis retinal. Photoisomerization of 11-cis to all-trans leads to structural changes in the protein of cytoplasmic loops, activating G-protein. Microbial rhodopsins are similar heptahelical membrane proteins that function as bacterial sensors, light-driven ion-pumps, or light-gated channels. They possess an all-trans retinal, and photoisomerization to 13-cis triggers structural changes in protein. Despite these similarities, there is no sequence homology between visual and microbial rhodopsins, and microbial rhodopsins do not activate G-proteins. In this study, new chimeric proton-pumping rhodopsins, proteorhodopsin (PR) and Gloeobacter rhodopsin (GR) were designed by replacing cytoplasmic loops with bovine Rh loops. Although G-protein was not activated by the PR chimeras, all 12 GR chimeras activated G-protein. The GR chimera containing the second cytoplasmic loop of bovine Rh did not activate G-protein. However, the chimera with a second and third double-loop further enhanced G-protein activation. Introduction of an E132Q mutation slowed the photocycle 30-fold and enhanced activation. The highest catalytic activity of the GR chimera was still 3,200 times lower than bovine Rh but only 64 times lower than amphioxus Go-rhodopsin. This GR chimera showed a strong absorption change of the amide-I band on a light-minus-dark difference FTIR spectrum which could represent a larger helical opening, important for G-protein activation. The light-dependent catalytic activity of this GR chimera makes it a potential optogenetic tool for enzymatic activation by light.

Highlights

Rhodopsin (Rh) is one of the G protein-coupled receptors (GPCRs) that has diverged into a photoreceptive protein in retinal visual cells [1,2,3,4,5,6]
We previously reported that the A178X mutant in the third loop of PR caused a 20-nm red shift in absorption and increased the pKa of the Schiff base counterion [38,39], which was fully reproduced in this study
We found an unusual volumedependent change in color of position 178, suggesting that the equilibrium of both PR protein conformations is dependent on the amino acid at that position

Summary

Introduction

Rhodopsin (Rh) is one of the G protein-coupled receptors (GPCRs) that has diverged into a photoreceptive protein in retinal visual cells [1,2,3,4,5,6]. We reported that chimeras of Natronomonas pharaonis SRII containing third loops of bovine Rh are able to activate G-protein [19]. This observation suggests that a common structural feature for light-induced activation among BR, SRII and bovine Rh exists in which the helix opening motion on the cytoplasmic side probably exposes the third loop to possible binding with G-protein. These chimeras have the potential to be tools for optogenetics [20,21]. Chimeric proton-pumping rhodopsins were designed using Proteorhodopsin (PR) from marine c-proteobacteria [24] and Gloeobacter rhodopsin (GR) from thylakoidless cyanobacteria to make new microbial rhodopsin chimeras with a higher G-protein activating function [25,26]

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