Ultrafast dynamics of biomolecules studied by vibrational spectroscopy on selectively isotope labeled proteins
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 addressed the nature and formation of the signaling state of Blue light using FAD (BLUF) photoreceptors using chemically and isotope labeled proteins. By 13C labeling of tryptophan residues in Slr1694 we experimentally identified for the first time a lightinduced extension of a beta sheet in the C-terminal region of the BLUF domain that involves the backbone a semi-conserved tryptophan. Its indol side-chain, which was previously suggested to be involved in conformational switching, 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. Modulation of the tyrosine redox potential by replacing it with 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 that will cover the complete timerange from femtoseconds to several microseconds 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 within the first nanoseconds, as well as structural processes taking place afterwards.
Publications
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(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
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(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
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(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
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(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
