Abstract
Enzyme rhodopsins, including cyclase opsins (Cyclops) and rhodopsin phosphodiesterases (RhoPDEs), were recently discovered in fungi, algae and protists. In contrast to the well-developed light-gated guanylyl/adenylyl cyclases as optogenetic tools, ideal light-regulated phosphodiesterases are still in demand. Here, we investigated and engineered the RhoPDEs from Salpingoeca rosetta, Choanoeca flexa and three other protists. All the RhoPDEs (fused with a cytosolic N-terminal YFP tag) can be expressed in Xenopus oocytes, except the AsRhoPDE that lacks the retinal-binding lysine residue in the last (8th) transmembrane helix. An N296K mutation of YFP::AsRhoPDE enabled its expression in oocytes, but this mutant still has no cGMP hydrolysis activity. Among the RhoPDEs tested, SrRhoPDE, CfRhoPDE1, 4 and MrRhoPDE exhibited light-enhanced cGMP hydrolysis activity. Engineering SrRhoPDE, we obtained two single point mutants, L623F and E657Q, in the C-terminal catalytic domain, which showed ~40 times decreased cGMP hydrolysis activity without affecting the light activation ratio. The molecular characterization and modification will aid in developing ideal light-regulated phosphodiesterase tools in the future.
Highlights
Academic Editors: Dmitry A.Rhodopsins are uniquely light-sensitive integral membrane proteins, sensing over a wide range of the visible spectrum, from UV to red light
We characterized the SrRhoPDE from Choanoflagellate Salpingoeca rosetta to be a blue light-stimulated phosphodiesterase that can degrade both cGMP
Alignment with SrRhoPDE showed that Cf RhoPDE1 and MrRhoPDE had a greater similarity to SrRhoPDE at 81% and 91%, while the AsRhoPDE had the lowest similarity at 49% (Table 2)
Summary
Rhodopsins are uniquely light-sensitive integral membrane proteins, sensing over a wide range of the visible spectrum, from UV to red light. The type I microbial rhodopsins were originally discovered to comprise seven transmembrane helices and function as ion channels or pumps, which directly convert light stimulation to a change in the electrochemical potential [1,2]. This enables the application of these light-sensitive proteins as optogenetic tools. ChR2 and its variants became powerful optogenetic tools in neuroscience [4,5,6,7] (ChR2) from the green alga Chlamydomonas reinhardtii were the first light-gated cation channels [1,3] discovered, enabling light-dependent depolarization of different cell types.
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