Functional dynamics of a single tryptophan residue in a BLUF protein revealed by fluorescence spectroscopy

Karadi, Kristof, Kapetanaki, Sofia M., Raics, Katalin, Pecsi, Ildiko, Kapronczai, Robert, Fekete, Zsuzsanna, Iuliano, James N., Tolentino Collado, Jinnette, Gil, Agnieszka A., Orban, Jozsef, Nyitrai, Miklos, Greetham, Gregory M., Vos, Marten H., Tonge, Peter J., Meech, Stephen R. ORCID: and Lukacs, Andras (2020) Functional dynamics of a single tryptophan residue in a BLUF protein revealed by fluorescence spectroscopy. Scientific Reports, 10. ISSN 2045-2322

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Blue Light Using Flavin (BLUF) domains are increasingly being adopted for use in optogenetic constructs. Despite this, much remains to be resolved on the mechanism of their activation. The advent of unnatural amino acid mutagenesis opens up a new toolbox for the study of protein structural dynamics. The tryptophan analogue, 7-aza-Trp (7AW) was incorporated in the BLUF domain of the Activation of Photopigment and pucA (AppA) photoreceptor in order to investigate the functional dynamics of the crucial W104 residue during photoactivation of the protein. The 7-aza modification to Trp makes selective excitation possible using 310 nm excitation and 380 nm emission, separating the signals of interest from other Trp and Tyr residues. We used Förster energy transfer (FRET) between 7AW and the flavin to estimate the distance between Trp and flavin in both the light- and dark-adapted states in solution. Nanosecond fluorescence anisotropy decay and picosecond fluorescence lifetime measurements for the flavin revealed a rather dynamic picture for the tryptophan residue. In the dark-adapted state, the major population of W104 is pointing away from the flavin and can move freely, in contrast to previous results reported in the literature. Upon blue-light excitation, the dominant tryptophan population is reorganized, moves closer to the flavin occupying a rigidly bound state participating in the hydrogen-bond network around the flavin molecule.

Item Type: Article
Uncontrolled Keywords: general ,/dk/atira/pure/subjectarea/asjc/1000
Faculty \ School: Faculty of Science > School of Chemistry
UEA Research Groups: Faculty of Science > Research Groups > Chemistry of Light and Energy
Faculty of Science > Research Groups > Centre for Photonics and Quantum Science
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Depositing User: LivePure Connector
Date Deposited: 25 Jan 2020 03:33
Last Modified: 09 Feb 2023 13:47
DOI: 10.1038/s41598-020-59073-5


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