Mechanism of Enzyme Rhodopsin Activation

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.

This has been explained in the summary above. The spectroscopic and biochemical characterization of the CaRhGC was our main achievement. CaRhGC can be purified as a protein that is biochemically and photochemically more stable than BeRhGC and is a much better suited for ultra fast spectroscopy and structural studies. RhGCs are the first characterized members of the large group of enzyme rhodopsins.

The most recent highlight and main break through of the project was the discovery of heterodimeric rhodopsin-cyclases (RhGCs) comprising a green absorbing RhGC monomer and a far-red absorbing RhGC monomer, named NeoR. The properties of NeoR are absolutely spectacular for the rhodopsin field because it absorbs maximally at 690 nm, the extinction coefficient is with 120000 M-1cm-1 three times higher than for most other rhodopsins, and the protein is highly fluorescence (QY=0.2) whereas all other rhodopsins are weakly or non fluorescent (QY < 0.0001).

We have studied the inner mechanics of the Cyclase and its activation and modulation by the different rhodopsins and have laid the basis for a general understanding of light activated enzymes when ever a rhodopsin module is involved.

The NeoR properties might be adaptable to other rhodopsins and opens the new research and application field of far-red Optogenetics which could be an add-on or an alternative for the visible Optogenetics used in the neurosciences so far.

Our goal for the whole project is a deep mechanistic understanding about structure and dynamics of RhGCs, about signal transmission from the rhodopsin to the cyclase and a wide optogenetic application in two component systems for sustained hyperpolarization at low light intensities.

Periodic Reporting for period 4 - MERA (Mechanism of Enzyme Rhodopsin Activation)

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