FG 526: Three blue light receptors - TP II: Biochemical and spectroscopic characterization of three blue light receptors with LOV and BLUF-domain-type chromophores

We have studied the photochemical processes that occur in the LOV1-domain of the blue-light receptor Phot1 from Chlamydomonas for the last three years. Although we gained very detailed insight in the structural basis and molecular mechanism of the photoreaction of the isolated LOV1-domain, the formation and structure of the biologically active signaling state is not yet clear. We propose to analyze the electron transfer process that has been assumed to occur in the triplet state of LOV1 (FMN-type) in more detail, and to study the subsequent formation of the signaling states in Phot1 A highly interdisciplinary approach combining molecular biology, biochemistry, spectroscopy, crystallography, and theory will be undertaken.
Second, we plan to study sensory blue light receptors containing the so-called BLUF domain as chromophores (FAD-type). We will express the AppA-Protein, which is involved in the regulation of genes for photosynthetic proteins in the purple bacterium R.sphaeroides, and PAC- that function as light-regulated adenylate cyclases in the flagellate E.gracilis. We will characterize the light activated enzymatic activities and try to determine the structures of both proteins in the dark form, and the light-activated state. We will study the primary photoprocesses by time and wavelength resolved UV-Vis and fluorescence spectroscopy, in particular in the ns time range. In this we face the challenge that the photoreceptors have very long cycle times (minutes), in some mutants the reaction is irreversible. The spectral and kinetic information will be combined with the structural data to describe in detail the physical and photochemical reactions involved in the formation of the signaling states.