Investigation of BLUF photochemistry by isotopic labeling of flavin cofactor and amino acid side chains
Final Report Abstract
Photosensory receptors are intriguing proteins for the study of biomolecular reactions since the photoinduced structural changes can be triggered with a flash of light and thus monitored with ultrafast time resolution. Still, data obtained by highly sensitive methods like vibrational spectroscopy is difficult to interpret molecularly because of ambiguity of assignments of strongly overlapping and convoluted signals. Here, we established a high performance and cost efficient strategy to introduce cofactor and amino acid specific isotope labels into flavoproteins to address nature and formation of the signaling state of Blue light using FAD (BLUF) photoreceptors. Particularly, the hydrogen bond network between the flavin chromophore and surrounding amino acids (glutamine, tryptophan, asparagine, histidine, tyrosine) and their ultrafast rearrangement upon formation of the biological signaling state were to be addressed. For this purpose essential genes for amino acid biosynthesis pathways have been deleted in an E. coli strain that contained a riboflavin transporter. The resulting strains are not only suitable to express flavoproteins efficiently by their ability to utilitze extracellular sources of riboflavin but can also be rendered flavin auxotrophic to allow simultaneous labeling of flavin and protein. The resulting amino acids auxotrophic strains for asparagine, histidine, tyrosine and tryptophan were used successfully for efficient and isotope scrambling free labeling. Unfortunately, even after introducing multiple deletions of the major glutamine degrading enzymes, labeling of glutamine did not succeed in a clean, scrambling free manner. Therefore, a major aspect of BLUF photoactivation, the conformation of a conserved and essential glutamine residue in light- and dark-adapted states could not be fully resolved. By labeling of tryptophan residues however, we experimentally identified for the first time a light-induced extension of a beta sheet in the C-terminal region of the Slr1694 BLUF. Its indol side-chain, which was previously suggested to be involved in conformational switching, in contrast, did not show any structural changes. The observed secondary structure change can explain the thermal stability of the signaling state and represent a key element in BLUF inter-/intramolecular signal transduction. Beyond isotope labeling, the strain collection described here also proved useful for introducing amino acid analogs. Modulation of the tyrosine redox potential by replacing it with a fluorinated analog affected both the speed of light-induced ultrafast electron transfer between tyrosine and flavin as well as the quantum yield of signaling state formation. Accordingly, we could demonstrate the relevance of electron transfer in BLUF signaling initiation. By investigation of the ultrafast dynamics of BLUF lacking the central glutamine we furthermore found strong indications that the formation of the neutral flavin semiquinone following the initial electron transfer drives the formation of the signaling state. Further ultrafast vibrational spectroscopy experiments are currently underway for various isotope labeled versions of Slr1694 that will allow us to pinpoint structural changes during the ultrafast formation of the signaling state.
Publications
- (2012) Redox Modulation of Flavin and Tyrosine Determines Photoinduced Proton-coupled Electron Transfer and Photoactivation of BLUF Photoreceptors. Journal of Biological Chemistry 287, 31725– 31738
Mathes T, van Stokkum IHM, Stierl M & Kennis JTM
(See online at https://doi.org/10.1074/jbc.M112.391896) - (2013) A Set of Engineered Escherichia coli Expression Strains for Selective Isotope and Reactivity Labeling of Amino Acid Side Chains and Flavin Cofactors. PLoS ONE 8, e79006
Mehlhorn J, Steinocher H, Beck S, Kennis JTM, Hegemann P & Mathes T
(See online at https://doi.org/10.1371/journal.pone.0079006) - (2015) Light-Induced Rearrangement of the β5 Strand in the BLUF Photoreceptor SyPixD (Slr1694). J. Phys. Chem. Lett. 6, 4749–4753
Mehlhorn J, Lindtner T, Richter F, Glaß K, Steinocher H, Beck S, Hegemann P, Kennis JTM & Mathes T
(See online at https://doi.org/10.1021/acs.jpclett.5b02245) - (2015) Photoinduced formation of flavin radicals in BLUF domains lacking the central glutamine. FEBS J 282, 3161–3174
Fudim R, Mehlhorn J, Berthold T, Weber S, Schleicher E, Kennis JTM & Mathes T
(See online at https://doi.org/10.1111/febs.13297) - (2015) Upgrading a microplate reader for photobiology and alloptical experiments. Photochem. Photobiol. Sci. 14, 270–279
Richter F, Scheib US, Mehlhorn J, Schubert R, Wietek J, Gernetzki O, Hegemann P, Mathes T & Möglich A
(See online at https://doi.org/10.1039/c4pp00361f)
