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A General Mechanism of Photoconversion of Green‐to‐Red Fluorescent Proteins Based on Blue and Infrared Light Reduces Phototoxicity in Live‐Cell Single‐Molecule Imaging
Author(s) -
Turkowyd Bartosz,
Balinovic Alexander,
Virant David,
Carnero Haruko G. Gölz,
Caldana Fabienne,
Endesfelder Marc,
Bourgeois Dominique,
Endesfelder Ulrike
Publication year - 2017
Publication title -
angewandte chemie international edition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.831
H-Index - 550
eISSN - 1521-3773
pISSN - 1433-7851
DOI - 10.1002/anie.201702870
Subject(s) - phototoxicity , fluorescence , chromophore , biophysics , threonine , chemistry , green fluorescent protein , serine , fluorescent protein , asparagine , photochemistry , residue (chemistry) , infrared , biochemistry , phosphorylation , amino acid , biology , optics , gene , physics , in vitro
Photoconversion of fluorescent proteins by blue and complementary near‐infrared light, termed primed conversion (PC), is a mechanism recently discovered for Dendra2. We demonstrate that controlling the conformation of arginine at residue 66 by threonine at residue 69 of fluorescent proteins from Anthozoan families (Dendra2, mMaple, Eos, mKikGR, pcDronpa protein families) represents a general route to facilitate PC. Mutations of alanine 159 or serine 173, which are known to influence chromophore flexibility and allow for reversible photoswitching, prevent PC. In addition, we report enhanced photoconversion for pcDronpa variants with asparagine 116. We demonstrate live‐cell single‐molecule imaging with reduced phototoxicity using PC and record trajectories of RNA polymerase in Escherichia coli cells.