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 10.1021/acs.jpclett.6b01172.s002 https://acs.figshare.com/articles/media/Fluorescence_from_Multiple_Chromophore_Hydrogen-Bonding_States_in_the_Far-Red_Protein_TagRFP675/3503204 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 <i>N</i>-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 S<sub>1</sub> 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. 2016-07-22 00:00:00 dynamic acylimine hydrogen bond conformers chromophore mKate vivo imaging applications TRF TagRFP 675 S 1 lifetime hydrogen bonds variant emission