TY - JOUR
T1 - Fluorescence from Multiple Chromophore Hydrogen-Bonding States in the Far-Red Protein TagRFP675
AU - Konold, Patrick E.
AU - Yoon, Eunjin
AU - Lee, Junghwa
AU - Allen, Samantha L.
AU - Chapagain, Prem P.
AU - Gerstman, Bernard S.
AU - Regmi, Chola K.
AU - Piatkevich, Kiryl D.
AU - Verkhusha, Vladislav V.
AU - Joo, Taiha
AU - Jimenez, Ralph
N1 - Publisher Copyright:
© 2016 American Chemical Society.
PY - 2016/8/4
Y1 - 2016/8/4
N2 - 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.
AB - 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.
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U2 - 10.1021/acs.jpclett.6b01172
DO - 10.1021/acs.jpclett.6b01172
M3 - Article
C2 - 27447848
AN - SCOPUS:84982817018
SN - 1948-7185
VL - 7
SP - 3046
EP - 3051
JO - Journal of Physical Chemistry Letters
JF - Journal of Physical Chemistry Letters
IS - 15
ER -