The cyclic nucleotides cAMP and cGMP are important second messengers that orchestrate fundamental cellular responses. We have characterized the rhodopsin guanylyl cyclase from Catenaria anguillulae (CaRhGC), which produces cGMP in response to green light with a light to dark activity ratio >1000. After light excitation the putative signaling state forms with τ = 31 ms and decays with τ = 570 ms. Mutations (up to 6) within the nucleotide binding site generate rhodopsin-adenylyl cyclases (CaRhACs) of which the double mutated YFP-CaRhAC (E497K/C566D) is the most suitable for rapid cAMP production in neurons. Furthermore, the crystal structure of the ligand-bound AC domain (2.25 Å) reveals detailed information about the nucleotide binding mode within this recently discovered class of enzyme rhodopsin. Both YFP-CaRhGC and YFP-CaRhAC are favorable optogenetic tools for non-invasive, cell-selective, and spatio-temporally precise modulation of cAMP/cGMP with light.
In addition, we have characterized two hetrodimeric RhGCs from Rhizoclosmatium globosum. RGC1 and RGC2 function as light-activated cyclases only upon heterodimerization with
RGC3 (NeoR). RGC1/2 utilize conventional green or blue-light-sensitive rhodopsins (λmax = 550 and 480 nm, respectively) responsible for light activation of the enzyme. The bistable NeoR is photoswitchable between a near-infrared sensitive (NIR, λmax = 690 nm) highly fluorescent state (QF = 0.2) and a UV-sensitive non-fluorescent state, thereby modulating the activity by NIR pre-illumination. No other rhodopsin has been reported so far to be functional as a heterooligomer, or as having such a long wavelength absorption or high fluorescence yield. Site-specific mutagenesis supports the idea that the unusual photochemical properties result from the rigidity of the retinal chromophore and a unique counterion triad and the exclusion of water. These findings substantially expand our understanding of the natural potential and limitations of spectral
tuning in rhodopsin photoreceptors.
Enzyme rhodopsins and in particular Rhodopsin-cyclases will be used for Optogenetic applications. This means the modulation of the important cellular second messengers cGMP and cAMP non-invasively with light. This could be of great relevance for the neurosciences as well as for cell biology and clinical research. The identification of NeoR expands the field of Optogenetics into the near infra red optical window where light penetrates animal tissues more efficiently than visible light and will open new windows for experimental and medical applications.
