Characterization and Modification of Light-Sensitive Phosphodiesterases from Choanoflagellates

Tian, Yuehui; Yang, Shang Fa; Nagel, Georg; Gao, Shiqiang

doi:10.3390/biom12010088

Cited by 5 publications

(4 citation statements)

References 34 publications

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“…We can find one candidate at the position of I119 in BR in the D-helix (TM4). Cf Rh-PDE3 and Cp Rh-PDE1 contain Asn at this position, while other Rh-PDEs possess Leu or Val (see below). − As this position is located near the β-ionone ring, the observed results were consistent with the general rule, where introduction of polar Asn near the β-ionone ring causes a spectral red-shift.…”

supporting

confidence: 60%

A Novel Color Switch of Microbial Rhodopsin

et al. 2023

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Function of animal and microbial rhodopsins starts by light absorption of the retinal chromophore. The absorption maximum wavelength (λ max ) of rhodopsins is determined by the energy gap between the electronically ground (S 0 ) and first excited (S 1 ) state of the retinal chromophore, and the color tuning mechanism is one of the central topics in rhodopsin research. "Color switches", color-determining residues, are red-and blue-shifting amino acids at the same position in two rhodopsins, whose exchange causes spectral blue-and red-shifts, respectively, in each rhodopsin. As mutation easily destroys elaborate chromophore−protein interactions, the known color switches in microbial rhodopsins are limited; the L/Q switch in C-helix (TM3), the A/TS switch in G-helix (TM7), and the G/P switch in F-helix (TM6). Here, we report a novel color switch of microbial rhodopsins, which is located in D-helix (TM4). In this color switch, the red-and blue-shifting amino acids are Asn (N) and Leu (L)/Ile (I), respectively. As Asn and Leu/Ile are polar and nonpolar amino acids, respectively, and the position is located near the β-ionone ring, the N/LI switch matches the general rule of color tuning by polarity. The N/LI switch is also useful for optogenetics, as many ion-transporting rhodopsins contain blue-shifting amino acids, such as L and I, at that position.

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confidence: 60%

A Novel Color Switch of Microbial Rhodopsin

et al. 2023

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“…8-nitro-cGMP) 37,38 , which in turn could activate PDE2 and PDE3, both of which are known to be upregulated by cGMP production to increase cAMP breakdown 39,40 . In addition, PDE isoforms directly activated by blue light have been reported in bacteria 41,42 , and blue-light modulation of AC activity has also been described [43][44][45][46] . We therefore set out to asses which stage in the biological process is most sensitive for blue light excitation.…”

Section: Discussionmentioning

confidence: 99%

‘Radical’ differences between two FLIM microscopes affect interpretation of cell signaling dynamics

Mukherjee,

Klarenbeek,

Oualid

et al. 2023

Preprint

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Emerging evidence suggests that cell signaling outcomes depend not only on the signal strength but also on its temporal progression. Our lab employs Fluorescence Lifetime Imaging of Resonance Energy Transfer (FLIM/FRET) biosensors to study intracellular signaling dynamics. We studied activation of β1 receptors by Isoproterenol, which triggers cAMP signaling via the G protein Gαs, using two different FLIM microscopes: a widefield frequency domain FLIM (fdFLIM) setup and a fast confocal Time Correlated Single Photon Counting (TCSPC) setup. When comparing results from each FLIM setup, unexpectedly we obtained distinctively different cAMP kinetics: fdFLIM recording of cAMP in HeLa and Cos7 cells yielded transient responses, reminiscent of rapid receptor desensitization, while TCSPC recordings exhibited sustained responses lasting over 30 minutes. We initially suspected phototoxicity due to the intense light locally in the laser focus spot in confocal microscopy to interfere with normal termination of signal transduction and set out to map photosensitive steps in the signaling cascade in detail. We find no evidence for light-sensitivity in either generation or breakdown of cAMP, but rather, our findings show that the kinetic differences are due to selective degradation of β1 agonists on the fdFLIM setup. Agonist degradation appeared due to the commercial FluoroBrite medium, even though this has been specifically advertised to lower phototoxicity and reduce autofluorescence. Mass spectrometry identified Folic acid, an undisclosed constituent of FluoroBrite, as the culprit leading to artifacts in fdFLIM measurements. In all, our study underscores the impact of subtle phototoxicity effects on experimental outcome, and it shows that in this case confocal TCSPC provides the more reliable data needed to study response kinetics. This work also emphasizes the it is crucial that scientific vendors fully disclose chemical formulations.

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“…Overall, type-1 pigments are the dominant contributors to marine phototrophy ( Casey et al, 2017 ; Larkum et al, 2018 ; Gómez-Consarnau et al, 2019 ). In addition, eukaryotic type-1 rhodopsins have been discovered which are intracellularly fused to an enzymatic domain and mediate light-driven enzyme activation (guanylyl cyclase, phosphodiesterase) or inhibition (guanylyl cyclase, based upon histidine kinase activity) ( Avelar et al, 2014 ; Lamarche et al, 2017 ; Luck et al, 2019 ; Mukherjee et al, 2019 ; Tsunoda et al, 2021 ; Broser, 2022 ; Tian et al, 2022 ). These pigments have been termed as enzyme-rhodopsins.…”

Section: Functional Diversity Phylogenymentioning

confidence: 99%

Rhodopsins: An Excitingly Versatile Protein Species for Research, Development and Creative Engineering

Grip

Ganapathy

2022

Front. Chem.

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The first member and eponym of the rhodopsin family was identified in the 1930s as the visual pigment of the rod photoreceptor cell in the animal retina. It was found to be a membrane protein, owing its photosensitivity to the presence of a covalently bound chromophoric group. This group, derived from vitamin A, was appropriately dubbed retinal. In the 1970s a microbial counterpart of this species was discovered in an archaeon, being a membrane protein also harbouring retinal as a chromophore, and named bacteriorhodopsin. Since their discovery a photogenic panorama unfolded, where up to date new members and subspecies with a variety of light-driven functionality have been added to this family. The animal branch, meanwhile categorized as type-2 rhodopsins, turned out to form a large subclass in the superfamily of G protein-coupled receptors and are essential to multiple elements of light-dependent animal sensory physiology. The microbial branch, the type-1 rhodopsins, largely function as light-driven ion pumps or channels, but also contain sensory-active and enzyme-sustaining subspecies. In this review we will follow the development of this exciting membrane protein panorama in a representative number of highlights and will present a prospect of their extraordinary future potential.

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Characterization and Modification of Light-Sensitive Phosphodiesterases from Choanoflagellates

Cited by 5 publications

References 34 publications

A Novel Color Switch of Microbial Rhodopsin

A Novel Color Switch of Microbial Rhodopsin

‘Radical’ differences between two FLIM microscopes affect interpretation of cell signaling dynamics

Rhodopsins: An Excitingly Versatile Protein Species for Research, Development and Creative Engineering

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