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