title: Single-fluorophore membrane transport activity sensors with dual-emission read-out.
abstract: We recently described a series of genetically encoded, single-fluorophore-based sensors, termed AmTrac and MepTrac, which monitor membrane transporter activity in vivo (De Michele et al., 2013). However, being intensiometric, AmTrac and Meptrac are limited in their use for quantitative studies. Here, we characterized the photophysical properties (steady-state and time-resolved fluorescence spectroscopy as well as anisotropy decay analysis) of different AmTrac sensors with diverging fluorescence properties in order to generate improved, ratiometric sensors. By replacing key amino acid residues in AmTrac we constructed a set of dual-emission AmTrac sensors named deAmTracs. deAmTracs show opposing changes of blue and green emission with almost doubled emission ratio upon ammonium addition. The response ratio of the deAmTracs correlated with transport activity in mutants with altered capacity. Our results suggest that partial disruption of distance-dependent excited-state proton transfer is important for the successful generation of single-fluorophore-based dual-emission sensors.
authors.affiliation: Department of Plant Biology, Carnegie Institution for Science, Stanford California, United States.
authors.forename: Cindy
authors.initials: C
authors.lastname: Ast
authors.affiliation: Institute of Biosciences and Bioresources, Italian National Research Council, Palermo, Italy.
authors.forename: Roberto
authors.initials: R
authors.lastname: De Michele
authors.affiliation: Department of Physical Chemistry, Institute of Chemistry, University of Potsdam, Potsdam, Germany.
authors.forename: Michael U
authors.initials: MU
authors.lastname: Kumke
authors.affiliation: Department of Plant Biology, Carnegie Institution for Science, Stanford California, United States.
authors.forename: Wolf B
authors.initials: WB
authors.lastname: Frommer
mesh_terms: D001692:Biological Transport; D015374:Biosensing Techniques / Q000379:methods*; D002462:Cell Membrane / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000032:analysis; D006801:Humans; D013050:Spectrometry, Fluorescence
chemical_list: D005456:Fluorescent Dyes
keywords: S. cerevisiae; biophysics; biosensor; cell biology; excited state proton transfer (ESPT); fluorescence; green fluorescent protein; structural biology; transporter