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

Authors
  • Karadi, Kristof1, 1
  • Kapetanaki, Sofia M.1, 1
  • Raics, Katalin1
  • Pecsi, Ildiko1
  • Kapronczai, Robert1
  • Fekete, Zsuzsanna1
  • Iuliano, James N.2
  • Collado, Jinnette Tolentino2
  • Gil, Agnieszka A.2
  • Orban, Jozsef1
  • Nyitrai, Miklos1, 1
  • Greetham, Greg M.3
  • Vos, Marten H.4
  • Tonge, Peter J.2
  • Meech, Stephen R.5
  • Lukacs, Andras1, 1
  • 1 University of Pécs, Pécs, 7624, Hungary , Pécs (Hungary)
  • 2 Stony Brook University, Stony Brook, NY, 11794-3400, USA , Stony Brook (United States)
  • 3 Central Laser Facility, Harwell Science and Innovation Campus, Didcot, Oxfordshire, UK , Didcot (United Kingdom)
  • 4 LOB, CNRS, INSERM, Ecole Polytechnique, Institut Polytechnique de Paris, Palaiseau, Cedex, 91128, France , Palaiseau (France)
  • 5 University of East Anglia, Norwich, NR4 7TJ, UK , Norwich (United Kingdom)
Type
Published Article
Journal
Scientific Reports
Publisher
Springer Nature
Publication Date
Feb 06, 2020
Volume
10
Issue
1
Identifiers
DOI: 10.1038/s41598-020-59073-5
Source
Springer Nature
License
Green

Abstract

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.

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