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

Angew Chem Int Ed Engl. 2017 Sep 11;56(38):11634-11639. doi: 10.1002/anie.201702870. Epub 2017 Jul 17.

Abstract

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.

Keywords: fluorescent proteins; live-cell microscopy; phototoxicity; primed conversion; single-molecule microscopy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Escherichia coli / cytology*
  • Escherichia coli / metabolism
  • Escherichia coli / radiation effects*
  • Green Fluorescent Proteins / chemistry
  • Green Fluorescent Proteins / metabolism*
  • Infrared Rays*
  • Light*
  • Luminescent Proteins / chemistry
  • Luminescent Proteins / metabolism*
  • Microbial Viability / radiation effects*
  • Microscopy, Fluorescence
  • Molecular Structure
  • Optical Imaging
  • Photochemical Processes / radiation effects
  • Red Fluorescent Protein
  • Single Molecule Imaging*

Substances

  • Luminescent Proteins
  • Green Fluorescent Proteins