Dataset Information

An engineered membrane-bound guanylyl cyclase with light-switchable activity.

ABSTRACT:

Background

Microbial rhodopsins vary in their chemical properties, from light sensitive ion transport to different enzymatic activities. Recently, a novel family of two-component Cyclase (rhod)opsins (2c-Cyclop) from the green algae Chlamydomonas reinhardtii and Volvox carteri was characterized, revealing a light-inhibited guanylyl cyclase (GC) activity. More genes similar to 2c-Cyclop exist in algal genomes, but their molecular and physiological functions remained uncharacterized.

Results

Chlamyopsin-5 (Cop5) from C. reinhardtii is related to Cr2c-Cyclop1 (Cop6) and can be expressed in Xenopus laevis oocytes, but shows no GC activity. Here, we exchanged parts of Cop5 with the corresponding ones of Cr2c-Cyclop1. When exchanging the opsin part of Cr2c-Cyclop1 with that of Cop5, we obtained a bi-stable guanylyl cyclase (switch-Cyclop1) whose activity can be switched by short light flashes. The GC activity of switch-Cyclop1 is increased for hours by a short 380 nm illumination and switched off (20-fold decreased) by blue or green light. switch-Cyclop1 is very light-sensitive and can half-maximally be activated by ~ 150 photons/nm² of 380 nm (~ 73 J/m²) or inhibited by ~ 40 photons/nm² of 473 nm (~ 18 J/m²).

Conclusions

This engineered guanylyl cyclase is the first light-switchable enzyme for cGMP level regulation. Light-regulated cGMP production with high light-sensitivity is a promising technique for the non-invasive investigation of the effects of cGMP signaling in many different tissues.

SUBMITTER: Tian Y

PROVIDER: S-EPMC8006352 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe green algae Chlamydomonas reinhardtii and Volvox carteri are important models for studying light perception and response, expressing many different photoreceptors. More than 10 opsins were reported in C. reinhardtii, yet only two-the channelrhodopsins-were functionally characterized. Characterization of new opsins would help to understand the green algae photobiology and to develop new tools for optogenetics.ResultsHere we report the characterization of a novel opsin family from these green algae: light-inhibited guanylyl cyclases regulated through a two-component-like phosphoryl transfer, called "two-component cyclase opsins" (2c-Cyclops). We prove the existence of such opsins in C. reinhardtii and V. carteri and show that they have cytosolic N- and C-termini, implying an eight-transmembrane helix structure. We also demonstrate that cGMP production is both light-inhibited and ATP-dependent. The cyclase activity of Cr2c-Cyclop1 is kept functional by the ongoing phosphorylation and phosphoryl transfer from the histidine kinase to the response regulator in the dark, proven by mutagenesis. Absorption of a photon inhibits the cyclase activity, most likely by inhibiting the phosphoryl transfer. Overexpression of Vc2c-Cyclop1 protein in V. carteri leads to significantly increased cGMP levels, demonstrating guanylyl cyclase activity of Vc2c-Cyclop1 in vivo. Live cell imaging of YFP-tagged Vc2c-Cyclop1 in V. carteri revealed a development-dependent, layer-like structure at the immediate periphery of the nucleus and intense spots in the cell periphery.ConclusionsCr2c-Cyclop1 and Vc2c-Cyclop1 are light-inhibited and ATP-dependent guanylyl cyclases with an unusual eight-transmembrane helix structure of the type I opsin domain which we propose to classify as type Ib, in contrast to the 7 TM type Ia opsins. Overexpression of Vc2c-Cyclop1 protein in V. carteri led to a significant increase of cGMP, demonstrating enzyme functionality in the organism of origin. Fluorescent live cell imaging revealed that Vc2c-Cyclop1 is located in the periphery of the nucleus and in confined areas at the cell periphery.