jz6b01172_si_004.mpg (6.2 MB)
Fluorescence from Multiple Chromophore Hydrogen-Bonding States in the Far-Red Protein TagRFP675
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posted on 2016-07-22, 00:00 authored by 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 JimenezFar-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.