Abstract
The rhodopsin-guanylyl cyclase from the nematophagous fungus Catenaria anguillulae belongs to a recently discovered class of enzymerhodopsins and may find application as a tool in optogenetics. Here the rhodopsin domain CaRh of the rhodopsin-guanylyl cyclase from Catenaria anguillulae was studied by absorption and emission spectroscopic methods. The absorption cross-section spectrum and excitation wavelength dependent fluorescence quantum distributions of CaRh samples were determined (first absorption band in the green spectral region). The thermal stability of CaRh was studied by long-time attenuation measurements at room temperature (20.5 °C) and refrigerator temperature of 3.5 °C. The apparent melting temperature of CaRh was determined by stepwise sample heating up and cooling down (obtained apparent melting temperature: 62 ± 2 °C). The photocycle dynamics of CaRh was investigated by sample excitation to the first inhomogeneous absorption band of the CaRhda dark-adapted state around 590 nm (long-wavelength tail), 530 nm (central region) and 470 nm (short-wavelength tail) and following the absorption spectra development during exposure and after exposure (time resolution 0.0125 s). The original protonated retinal Schiff base PRSBall-trans in CaRhda photo-converted reversibly to protonated retinal Schiff base PRSBall-trans,la1 with restructured surroundings (CaRhla1 light-adapted state, slightly blue-shifted and broadened first absorption band, recovery to CaRhda with time constant of 0.8 s) and deprotonated retinal Schiff base RSB13-cis (CaRhla2 light-adapted state, first absorption band in violet to near ultraviolet spectral region, recovery to CaRhda with time constant of 0.35 s). Long-time light exposure of light-adapted CaRhla1 around 590, 530 and 470 nm caused low-efficient irreversible degradation to photoproducts CaRhprod. Schemes of the primary photocycle dynamics of CaRhda and the secondary photocycle dynamics of CaRhla1 are developed.
Highlights
The nematophagous fungus Catenaria anguillulae is a facultative endoparasite of free living and plant parasitic nematodes ([1,2] and references therein)
The rhodopsin domain CaRh of the rhodopsin-guanylyl cyclase CaRhGC from Catenaria anguillulae was studied by absorption and emission spectroscopic methods
Its photophysical behavior was compared with that of BeRh, the rhodopsin domain of the rhodopsin-guanylyl cyclase BeRhGC from the aquatic fungus Blastocladiella emersonii. Both rhodopsin-guanylyl cyclases belong to the class of emzymerhodopsins in microbial organisms
Summary
The nematophagous fungus Catenaria anguillulae is a facultative endoparasite of free living and plant parasitic nematodes ([1,2] and references therein). Searches of the genome assembly of Catenaria anguillulae found the presence of a rhodopsin-guanylyl cyclase gene fusion [3]. This guanylyl cyclase opsin, named CaCyclOp in [4], was expressed and preliminarily characterized respective cGMP (cyclic guanosine monophosphate) production in Xenopus oocytes [4]. A more extensive study of rhodopsin-cyclases for light-induced cGMP and cAMP (cyclic adenosine monophosphate) production was carried out with the related rhodopsin-guanylyl cyclase from Blastocladiella emersonii BeRhGC [4,5]. Recombinant full-length BeRhGC was thermally unstable and light-induced cyclase activity could not be measured. The recombinant rhodopsin fragment BeRh was characterized respective its photophysical and photochemical properties [5,6]. The full-length protein BeRhGC could be functionally expressed in hippocampal neurons, making it usable to optogenetic applications [5]
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