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  3. Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo

Small fluorescence-activating and absorption-shifting tag for tunable protein imaging in vivo

  • Proc Natl Acad Sci U S A. 2016 Jan 19;113(3):497-502. doi: 10.1073/pnas.1513094113.
Marie-Aude Plamont 1,  Emmanuelle Billon-Denis 1,  Sylvie Maurin 1,  Carole Gauron 2,  Frederico M Pimenta 1,  Christian G Specht 3,  Jian Shi 1,  Jérôme Quérard 1,  Buyan Pan 1,  Julien Rossignol 1,  Karine Moncoq 4,  Nelly Morellet 5,  Michel Volovitch 6,  Ewen Lescop 5,  Yong Chen 1,  Antoine Triller 3,  Sophie Vriz 7,  Thomas Le Saux 1,  Ludovic Jullien 8,  Arnaud Gautier 8
Affiliations

Affiliations

  • 1 Department of Chemistry, École Normale Supérieure, Paris Sciences et Lettres (PSL) Research University, F-75005 Paris, France; Sorbonne Universités, Université Pierre et Marie Curie, UMR 8640 PASTEUR, F-75005 Paris, France; CNRS, UMR 8640 PASTEUR, F-75005 Paris, France;
  • 2 Centre for Interdisciplinary Research in Biology, CNRS UMR 7241, INSERM U1050, Labex MemoLife, Paris Sciences et Lettres (PSL) Research University/Collège de France, F-75005 Paris, France;
  • 3 École Normale Supérieure, Institut de Biologie de l'École Normale Supérieure (IBENS), CNRS UMR 8197, INSERM U1024, Paris Sciences et Lettres (PSL) Research University, F-75005 Paris, France;
  • 4 Laboratoire de Biologie Physico-Chimique des Protéines Membranaires, Institut de Biologie Physico-Chimique, CNRS UMR 7099, Université Paris Diderot, Sorbonne Paris Cité, Paris Sciences et Lettres (PSL) Research University, F-75005 Paris, France.
  • 5 Institut de Chimie des Substances Naturelles, CNRS UPR 2301, Université Paris-Saclay, 91198 Gif-sur-Yvette, France;
  • 6 Centre for Interdisciplinary Research in Biology, CNRS UMR 7241, INSERM U1050, Labex MemoLife, Paris Sciences et Lettres (PSL) Research University/Collège de France, F-75005 Paris, France; École Normale Supérieure, Institut de Biologie de l'École Normale Supérieure (IBENS), CNRS UMR 8197, INSERM U1024, Paris Sciences et Lettres (PSL) Research University, F-75005 Paris, France;
  • 7 Centre for Interdisciplinary Research in Biology, CNRS UMR 7241, INSERM U1050, Labex MemoLife, Paris Sciences et Lettres (PSL) Research University/Collège de France, F-75005 Paris, France; Université Paris Diderot Sorbonne Paris Cité, 75205 Paris, France.
  • 8 Department of Chemistry, École Normale Supérieure, Paris Sciences et Lettres (PSL) Research University, F-75005 Paris, France; Sorbonne Universités, Université Pierre et Marie Curie, UMR 8640 PASTEUR, F-75005 Paris, France; CNRS, UMR 8640 PASTEUR, F-75005 Paris, France; arnaud.gautier@ens.fr ludovic.jullien@ens.fr.
PMID: 26711992 DOI: 10.1073/pnas.1513094113
Abstract

This paper presents Yellow Fluorescence-Activating and absorption-Shifting Tag (Y-FAST), a small monomeric protein tag, half as large as the green Fluorescent protein, enabling fluorescent labeling of proteins in a reversible and specific manner through the reversible binding and activation of a cell-permeant and nontoxic fluorogenic ligand (a so-called fluorogen). A unique fluorogen activation mechanism based on two spectroscopic changes, increase of fluorescence quantum yield and absorption red shift, provides high labeling selectivity. Y-FAST was engineered from the 14-kDa photoactive yellow protein by directed evolution using yeast display and fluorescence-activated cell sorting. Y-FAST is as bright as common fluorescent proteins, exhibits good photostability, and allows the efficient labeling of proteins in various organelles and hosts. Upon fluorogen binding, fluorescence appears instantaneously, allowing monitoring of rapid processes in near real time. Y-FAST distinguishes itself from other tagging systems because the fluorogen binding is highly dynamic and fully reversible, which enables rapid labeling and unlabeling of proteins by addition and withdrawal of the fluorogen, opening new exciting prospects for the development of multiplexing imaging protocols based on sequential labeling.

Keywords

directed evolution; fluorescence imaging; fluorogenic ligand.

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