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 - Funding Information:
This work was supported by the NSF Physics Frontier Center at JILA and the National Institutes of Health (GM105997 and GM108579 to V.V.V and SC3GM096903 to P.P.C.). S.L.A. was supported by the University of Colorado Molecular Biophysics Training Grant (T32 GM065103). T.J. acknowledges the financial support by the Global Research Laboratory Program (2009-00439) through the National Research Foundation of Korea. R.J. is a staff member in the Quantum Physics Division of the National Institute of Standards and Technology (NIST). Certain commercial equipment, instruments, or materials are identified in this paper in order to specify the experimental procedure adequately. Such identification is not intended to imply recommendation or endorsement by the NIST, nor is it intended to imply that the materials or equipment identified arenecessarily the best available for the purpose.
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 -