Functional characterization of novel rhodopsins of Chlamydomonas and other algae

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During the current funding period we were able to characterize a number of so far non described rhodopsins of green algae. Most efforts we have invested in the characterization of rhodopsin domain that is part of the novel Histidin-Kinase Rhodopsin (HKR), a family that we recently discovered in Chlamydomonas. This protein is an UVA-receptor (λmax = 380 nm). After UV-light absorption it is converted into the stable Metaform Rh-Bl (λmax = 490 nm) and the reaction is reversed by blue light. Chromophore dynamics and photochemistry were studied by time resolved UV/Vis and Resonance Raman spectroscopy. In parallel we have characterized a Channelrhodopsin of the halotolerant alga Dunaliella salina, which exclusively conducts H+, and a light driven proton pump of Chlorella vulgaris. The Chlorella protein is of high interest for optogenetic applications. During the funding period we are applying for we would like to continue the characterization of the HKRs on a molecular level in vitro and in vivo. We will undertake efforts to functionally express the rhodopsins of HKR2 and HKR4 of Chlamydomonas and the only HKR of Ostreococcus to study spectral properties and photoreactions. We will try to express the full-length proteins and to biochemically characterize the signaling process including light regulated cyclase activity. We will delete the genes by homologous recombination and characterize the deletion strains by biophysical and physiological methods. As a side project we will continue the characterization of the only rhodopsins of the cryophilic alga Fragilariopsis cylindrus and the green alga Chlorella vulgaris.