Analysis and engineering of natural photoreceptors to Light-manipulate cyclic nucleotides, Ca2+ and membrane voltage in animal cells

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The cloning of a flavoprotein from the flagellate Euglena gracilis – Photoactivated Adenylyl Cyclase (PAC) – initiated the use of a new optogenetic tool, as light-induced production of cAMP by purified PACα2β2 was shown. We expressed PACα and PACβ separately in animal cells and showed fast and reversible light-induced [cAMP] increase with both, although 100-fold more effective with PACα. We intend to mutate PACα and PACβ to study the function of important amino acids and the interaction of PACα with PACβ. Recently we showed by experiments with mutated PACα that it functions as a PACα2 dimer. We will closely collaborate with collaborators working on the structure of blue-light receptors (project 6). We aim to convert PAC into a photo-activated guanylyl cyclase (PGC), a new tool for our collaborators in the research group, working with model organisms (projects 1, 5, 7). We plan to localize PAC to specific membrane sites by fusing it to different membrane proteins and make it available – after functional tests – to our collaborators. We propose to express microbial type rhodopsins (the light-activated Cl- pump halorhodopsin and the light-gated cation channel channelrhodopsin-2) as fragments, split into two protein moieties and to test functional reconstitution in the membrane. Such constructs, under the control of two different promoters, will enable more cellspecific expression, especially in the nervous system of Drosophila, C. elegans, and zebrafish. We will insert fluorescent proteins (FP) into interhelical loops of rhodopsins to confirm these loops and to enhance reconstitution of a functional rhodopsin from two fragments, split in the middle of the FP. We will collaborate with project 2 where the interaction of rhodopsin fragments is studied on supported membranes with surfaceenhanced spectroscopic techniques.