FOR 526: Blaulicht-sensitive Photorezeptoren
Zusammenfassung der Projektergebnisse
Flavin photobiology and photochemistry has been deeply investigated in the 60ies and 70ies of the last century. However, with the molecular identification of flavins as the light-absorbing chromophores in blue light-sensitive biological photoreceptors, flavin photochemistry has seen a revival. This can clearly be ascribed to three types of biological sensors with very different photo-reactivities: These are Cryptochromes with photolyase-akin electron transfer, LOV-proteins forming C(4a)-flavin-cysteinyl adduct formation, and BLUF-proteins that undergo thorough hydrogen bond rearrangement after light stimulation. This astonishing variability in photochemical processes has gained high interest in the scientific community; in Germany most of these on going activities have been bundled through a joint research project, FOR526 funded by the German Research Association (DFG). By this, blue light photoreceptor research has received special support over a total of six years for 12 groups, tightly working together in biophysical, chemical, biochemical and microbiological approaches. As a documentation for this joint research, the special issue of Photochemistry and Photobiology compiles manuscripts from these collaborating groups with major emphasis on the primary reactions of LOV- and BLUF-proteins. The work yielded deeper insight into the photo-catalyzed processes on isolated chromophores and chemically modified derivatives, and into the reaction pathways of the flavin photoreceptors. Experimental spectroscopic work has been supported by simulations from theoretical chemists and by strong contributions from protein crystallographers. As the flavin photochemistry is often connected with intermediate single electron transfer and radical formation, the contribution of spectroscopic methods, especially EPR-studies, was paramount for these projects. For the LOV-proteins the radical mechanism of photoadduct formation could be documented. FTIR-studies on LOV-domains with µs time resolution identified late photocycle intermediates and conformational changes within sensor and effector domains. LOV domain photochemistry is now well understood to a large extent, allowing the LOV domains to serve as model systems for a number of fundamental studies. The BLUF proteins (blue light sensors using FAD) still represent an enigmatic system. Less distributed in living organisms, some of their light induced reactions and their physiological functions are still under debate. Clearly so, as blue light irradiation causes simply a small bathochromic shift of the chromophore’s absorption maximum by 10 to 14 nm. Although this spectral change could be assigned to a re-organization of the hydrogen bonding network complexing the chromophore, its relevance to physiological responses and signal transduction pathway with the protein is far from being understood. Photocycles for several BLUF domains and also, for the first time, of a BLUF-Effector protein, BlrP, have been worked out and relevant dynamics parameters have been determined by absorption and vibrational spectroscopy. Ultra fast studies carried our in collaboration with the Kennis group in Amsterdam identified an early radical anion as an early reaction intermediate on ps time scale. New crystal structures were determined for AppA-BLUF and the intact photoreceptor BlrP1 consisting of a BLUF sensing domain and an EAL c-di-GMP phosphodiesterase output domain revealing new principles for the activation of effecter domains. Based on structural information and quantum mechanical simulations, models for the dark and light states of the BLUF domain were developed proposing that photon absorption by the flavin results in a tautomerization and rotation of the Gln side chain that interacts with the flavin cofactor. These early events cause alterations in the hydrogen bond network in the core of the photoreceptor domain as observed in numerous spectroscopic experiments. Light-induced isomerization and/or tautomerization of an amino acid residues seem to represent novel principles for photoreceptor activation. As mentioned already above, the light-regulated enzyme activities fused to LOV and BLUF-domains have surely attracted many scientists. Initially identified as naturally existing proteins (where e.g., histidine kinases, AMP cyclases, and c-di-GMP-modifying enzymes (phosphodiesterase and diguanylate cyclases are the major representatives), the utility of the chromophore domain has rapidly been recognized as an option for the generation of artificial, non-naturally occurring proteins. For the photoactivatable Rac1, a tandem of LOV2 and Rac1 designed by the Klaus Hahns group, the structure has been solved which provides insight into a synthetic light-activated protein. Also the combination of a LOV-domain and the trp-repressor (exemplifying light-induced DNA binding) stands for a new innovative direction of Blue light receptor research. For the photoactivated cyclase (PAC) from Euglena gracilis, a protein originally discovered by Iseki and colleagues, we demonstrated together with external collaborators PAC expression in neuronal cells and regulation of cellular cAMP levels. Though not covered by articles in this issue, the cryptochromes were an important fraction of the collaborative FOR work. Electron transfer-driven proton migration was demonstrated by FTIR for Arabidopsis Cry1. A DASH-type cryptochrome has been cocrystallized together with a repaired CPD- lesion in single-stranded DNA. Moreover, binding and repair of CPD-lesions in loop-structured duplex DNA by DASH-type cryptochrome has been manisfested. The group also unraveled the photoreduction of 5,10-methenyl-tetrahydrofolate antenna cofactor of DNA-photolyase and DASH- cryptochromes to 5,10-methylene-tetrahydrofolate. This highlights the presence of a third electron transfer pathway in members of the photolyase / cryptochrome family. For Drosophila Cryptochrome, dCRY, it was shown that oxidized FAD is converted in the light to an anionic FAD°- radical in contrast to plant CRYs which form the neutral FADH° radicals. Purified dCRY is partially loaded with 5,10-Methenyltetrahydrofolat (MTHF) suggesting that dCRY binds MTHF as a second (light-harvesting ?) chromophore in vivo. The FOR526, which focused on primary photochemical processes, has been officially closed almost a year ago after our final FOR conference at Frauenchiemsee but some of the graduate students continued the work due to a late start or maternity leave. The work on blue light photoreceptors is continuing in terms of a new research consortium FOR1279. Within this new constellation the research topic is the sensor-receptor coupling. The basis has been laid by studies on photo-activatable Rac and light-activatable cyclases (euPAC and bPAC) etc. The new network is also approaching basic biological principles but will also serve optogenetic application.
Projektbezogene Publikationen (Auswahl)
- (2002) First evidence for phototropin-like blue-light receptors in prokaryots. Biophys. J. 82 2627-2634
Losi, A., Polverini, E., Quest, B. and Gärtner, W.
- (2006) Hydrogen bond switching via a radical pair mechanism in a flavinbinding photoreceptor. PNAS 103, 10895-1090
Gauden, M., Key, J.M., van Stokkum, I.H.M., Luehrs, D.C., van Grondelle, R., Hegemann, P. and Kennis, J.T.M.
- (2006) ‘Blue-Light-Ind uced Changes in Arabidopsis Cryptochrome 1 Probed by FTIR Difference Spectroscopy’, Biochemistry 45, 2472- 2479
Kottke, T., Ahmad, M., Batschauer, A., & Heberle, J.
- (2007) A novel Photoreaction Mechanism for the circadian Blue-Light Photoreceptor Drosophila Cryptochrome. JBC 282, 13011-13021
Berndt A., Kottke T., Breitkreuz H., Dvorsky R., Hennig S., Alexander M. and Wolf E.
- (2007) In vivo fluorescence without oxygen: Novel fluorescence reporters for anaerobic systems. Nature Biotechnology 25 443-445
Drepper, T., Eggert, T., Circolone, F., Heck, A., Krauß, U., Guterl, J.-K., Wendorff, M., Losi, A, Gärtner, W., Jaeger, K.-E.
- (2008) Fast manipulation of cellular cAMP level by light in vivo. Nature Methods 4, 39-42
S. Schröder-Lang, M. Schwärzel, R. Seifert, T. Strünker, S. Kateriya, J. Looser, M. Watanabe, U.B. Kaupp, P. Hegemann, G. Nagel
- (2008) Fluorescence spectroscopic Characterization of the circadian blue-light Photoreceptor Cryptochrome from Drosophila melanogaster. Chemical Physics 28; 352: 35-47
Shirdel J., Zirak P., Penzkofer A., Breitkreuz H. and Wolf E.
- (2008) Recognition and repair of UV-lesions in loop structures of duplex DNA by DASH-type cryptochrome. Proc. Natl. Acad. Sci. USA 105, 21023-21027
Pokorny R., Klar T., Hennecke U., Carell T., Batschauer A., Essen L.-O.
- (2008): Molecular models predict light-induced glutamine tautomerization in BLUF photoreceptors. Biophys. J. 94: 3872- 3879
Domratcheva, T.; Grigorenko, B. L.; Schlichting, I.; Nemukhin, A. V.
- (2009) A genetically encoded photoactivatable Rac controls the motility of living cells. Nature 461, 104- 108
Wu, Y.I., Frey, D., Lungu, O.I., Jaehrig, A., Schlichting, I., Kuhlmann, B., Hahn, K.M.
- (2009) Photorededuction of the folate cofactor in members of the photolyase family. J. Biol. Chem. 284, 21670-21683
Moldt J., Pokorny R., Orth C., Linne U., Geisselbrecht Y., Marahiel M.A., Essen L.-O., Batschauer A.
- (2009) Structure and Mechanism of a Bacterial Light-Regulated Cyclic Nucleotide Phosphodiesterase. Nature, 459: 1015-1018
Barends, T.R.M., Hartmann, E., Griese, J.J., Beitlich, T., Kirienko, N.V., Ryjenkov, D.A., Reinstein, J., Shoeman, R.L., Gomelsky, M., and Schlichting, I.
- (2009)‘Time-Resolved Fourier Transform Infrared Study on Photoadduct Formation and Secondary Structural Changes within the Phototropin LOV Domain ’ Biophys. J. 96 , 1462-1470
Pfeifer, A., Majerus, T., Zikihara, K., Matsuoka, D., Tokutomi, S., Heberle, J., & Kottke, T.
- (2010) Absorption and emission spectroscopic characterisation of lumichrome in aqueous solutions, Photochem. Photobiol.
Tyagi, A. and Penzkofer, A.
(Siehe online unter https://doi.org/10.1111/j.1751-1097.2010.00836.x) - (2010) Dependence of the absorption and emission behaviour of lumiflavin in aqueous solution, J. Photochem. Photobiol. A: Chem. 215 108-117
Tyagi, A. and Penzkofer, A.
