Fluorescence from Multiple Chromophore Hydrogen-Bonding States in the Far-Red Protein TagRFP675

J Phys Chem Lett. 2016 Aug 4;7(15):3046-51. doi: 10.1021/acs.jpclett.6b01172. Epub 2016 Jul 27.


Far-red fluorescent proteins are critical for in vivo imaging applications, but the relative importance of structure versus dynamics in generating large Stokes-shifted emission is unclear. The unusually red-shifted emission of TagRFP675, a derivative of mKate, has been attributed to the multiple hydrogen bonds with the chromophore N-acylimine carbonyl. We characterized TagRFP675 and point mutants designed to perturb these hydrogen bonds with spectrally resolved transient grating and time-resolved fluorescence (TRF) spectroscopies supported by molecular dynamics simulations. TRF results for TagRFP675 and the mKate/M41Q variant show picosecond time scale red-shifts followed by nanosecond time blue-shifts. Global analysis of the TRF spectra reveals spectrally distinct emitting states that do not interconvert during the S1 lifetime. These dynamics originate from photoexcitation of a mixed ground-state population of acylimine hydrogen bond conformers. Strategically tuning the chromophore environment in TagRFP675 might stabilize the most red-shifted conformation and result in a variant with a larger Stokes shift.

Publication types

  • Letter

MeSH terms

  • Fluorescence
  • Hydrogen Bonding
  • Luminescent Proteins / chemistry*
  • Molecular Conformation
  • Molecular Dynamics Simulation


  • Luminescent Proteins
  • red fluorescent protein