Report
Teaser, summary, work performed and final results
Periodic Reporting for period 2 - MERA (Mechanism of Enzyme Rhodopsin Activation)
Teaser
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...
Summary
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 Ï„ = 31 ms and decays with Ï„ = 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 Ã…) 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.
Our goal is the in depth characterization of Rhodopsin-cyclases by application of a number of biophysical and biophysical techniques. Our intention is to dissect the signal transduction pathway into a number of molecular events starting from the light absorption to the full activation of the protein and the detailed reaction steps of catalysis during GTP to cGMP conversion.
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 overall objective is a detailed molecular and mechanistic understanding of rhodopsin cyclases as unique examples of membrane bound photoactivated enzymes.
Work performed
This has been explained in the summary above. The spectroscopic and biochemical characterization of the CaRhGC is 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.
Final results
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.