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Article Dans Une Revue Scientific Reports Année : 2020

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

Kristof Karadi
  • Fonction : Auteur
Sofia Kapetanaki
  • Fonction : Auteur
Katalin Raics
  • Fonction : Auteur
Ildiko Pecsi
  • Fonction : Auteur
Robert Kapronczai
  • Fonction : Auteur
Zsuzsanna Fekete
  • Fonction : Auteur
James Iuliano
  • Fonction : Auteur
Jinnette Tolentino Collado
  • Fonction : Auteur
Agnieszka Gil
  • Fonction : Auteur
Jozsef Orban
  • Fonction : Auteur
Greg Greetham
  • Fonction : Auteur
Marten H. Vos
Peter Tonge
  • Fonction : Auteur
Stephen Meech
Andras Lukacs

Résumé

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 darkadapted 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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Dates et versions

hal-02469113 , version 1 (24-11-2020)

Identifiants

Citer

Kristof Karadi, Sofia Kapetanaki, Katalin Raics, Ildiko Pecsi, Robert Kapronczai, et al.. Functional dynamics of a single tryptophan residue in a BLUF protein revealed by fluorescence spectroscopy. Scientific Reports, 2020, 10, pp.2061. ⟨10.1038/s41598-020-59073-5⟩. ⟨hal-02469113⟩
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