title: Generation of Fluorogen-Activating Designed Ankyrin Repeat Proteins (FADAs) as Versatile Sensor Tools.
abstract: Fluorescent probes constitute a valuable toolbox to address a variety of biological questions and they have become irreplaceable for imaging methods. Commonly, such probes consist of fluorescent proteins or small organic fluorophores coupled to biological molecules of interest. Recently, a novel class of fluorescence-based probes, fluorogen-activating proteins (FAPs), has been reported. These binding proteins are based on antibody single-chain variable fragments and activate fluorogenic dyes, which only become fluorescent upon activation and do not fluoresce when free in solution. Here we present a novel class of fluorogen activators, termed FADAs, based on the very robust designed ankyrin repeat protein scaffold, which also readily folds in the reducing environment of the cytoplasm. The FADA generated in this study was obtained by combined selections with ribosome display and yeast surface display. It enhances the fluorescence of malachite green (MG) dyes by a factor of more than 11,000 and thus activates MG to a similar extent as FAPs based on single-chain variable fragments. As shown by structure determination and in vitro measurements, this FADA was evolved to form a homodimer for the activation of MG dyes. Exploiting the favorable properties of the designed ankyrin repeat protein scaffold, we created a FADA biosensor suitable for imaging of proteins on the cell surface, as well as in the cytosol. Moreover, based on the requirement of dimerization for strong fluorogen activation, a prototype FADA biosensor for in situ detection of a target protein and protein-protein interactions was developed. Therefore, FADAs are versatile fluorescent probes that are easily produced and suitable for diverse applications and thus extend the FAP technology.
authors.affiliation: Department of Biochemistry, University of Zurich, CH-8057, Zurich, Switzerland.
authors.forename: Marco
authors.initials: M
authors.lastname: Schütz
authors.affiliation: Department of Biochemistry, University of Zurich, CH-8057, Zurich, Switzerland.
authors.forename: Alexander
authors.initials: A
authors.lastname: Batyuk
authors.affiliation: Department of Biochemistry, University of Zurich, CH-8057, Zurich, Switzerland.
authors.forename: Christoph
authors.initials: C
authors.lastname: Klenk
authors.affiliation: Department of Biochemistry, University of Zurich, CH-8057, Zurich, Switzerland.
authors.forename: Lutz
authors.initials: L
authors.lastname: Kummer
authors.affiliation: Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Seymour
authors.initials: S
authors.lastname: de Picciotto
authors.affiliation: Department of Chemistry and Applied Biosciences, ETH Zurich, CH-8093, Zurich, Switzerland.
authors.forename: Basri
authors.initials: B
authors.lastname: Gülbakan
authors.affiliation: Department of Biochemistry, University of Zurich, CH-8057, Zurich, Switzerland.
authors.forename: Yufan
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Department of Biochemistry, University of Zurich, CH-8057, Zurich, Switzerland.
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Newby
authors.affiliation: Department of Biochemistry, University of Zurich, CH-8057, Zurich, Switzerland.
authors.forename: Franziska
authors.initials: F
authors.lastname: Zosel
authors.affiliation: Department of Biochemistry, University of Zurich, CH-8057, Zurich, Switzerland.
authors.forename: Jendrik
authors.initials: J
authors.lastname: Schöppe
authors.affiliation: Department of Biochemistry, University of Zurich, CH-8057, Zurich, Switzerland.
authors.forename: Erik
authors.initials: E
authors.lastname: Sedlák
authors.affiliation: Department of Biochemistry, University of Zurich, CH-8057, Zurich, Switzerland.
authors.forename: Peer R E
authors.initials: PRE
authors.lastname: Mittl
authors.affiliation: Department of Chemistry and Applied Biosciences, ETH Zurich, CH-8093, Zurich, Switzerland.
authors.forename: Renato
authors.initials: R
authors.lastname: Zenobi
authors.affiliation: Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: K Dane
authors.initials: KD
authors.lastname: Wittrup
authors.affiliation: Department of Biochemistry, University of Zurich, CH-8057, Zurich, Switzerland. Electronic address: plueckthun@bioc.uzh.ch.
authors.forename: Andreas
authors.initials: A
authors.lastname: Plückthun
mesh_terms: D017089:Ankyrin Repeat; D015374:Biosensing Techniques / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D012394:Rosaniline Dyes / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D012394:Rosaniline Dyes; C005095:malachite green
keywords: biosensors; designed ankyrin repeat proteins; directed evolution; fluorogen-activating proteins; protein engineering