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

Patrick E. Konold, Eunjin Yoon, Junghwa Lee, Samantha L. Allen, Prem P. Chapagain, Bernard S. Gerstman, Chola K. Regmi, Kiryl D. Piatkevich, Vladislav V. Verkhusha, Taiha Joo, Ralph Jimenez

Research output: Contribution to journalArticlepeer-review

10 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)3046-3051
Number of pages6
JournalJournal of Physical Chemistry Letters
Issue number15
StatePublished - Aug 4 2016

ASJC Scopus subject areas

  • Materials Science(all)
  • Physical and Theoretical Chemistry


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