6 research outputs found
Hydrogen Bond Flexibility Correlates with Stokes Shift in mPlum Variants
Fluorescent proteins have revolutionized
molecular biology research
and provide a means of tracking subcellular processes with extraordinary
spatial and temporal precision. Species with emission beyond 650 nm
offer the potential for deeper tissue penetration and lengthened imaging
times; however, the origin of their extended Stokes shift is not fully
understood. We employed spectrally resolved transient grating spectroscopy
and molecular dynamics simulations to investigate the relationship
between the flexibility of the chromophore environment and Stokes
shift in mPlum. We examined excited state solvation dynamics in a
panel of strategic point mutants of residues E16 and I65 proposed
to participate in a hydrogen-bonding interaction thought responsible
for its red-shifted emission. We observed two characteristic relaxation
constants of a few picoseconds and tens of picoseconds that were assigned
to survival times of direct and water-mediated hydrogen bonds at the
16-65 position. Moreover, variants of the largest Stokes shift (mPlum,
I65V) exhibited significant decay on both time scales, indicating
the bathochromic shift correlates with a facile switching between
a direct and water-mediated hydrogen bond. This dynamic model underscores
the role of environmental flexibility in the mechanism of excited
state solvation and provides a template for engineering next-generation
red fluorescent proteins
High-Precision Megahertz-to-Terahertz Dielectric Spectroscopy of Protein Collective Motions and Hydration Dynamics
The
low-frequency collective vibrational modes in proteins as well
as the protein–water interface have been suggested as dominant
factors controlling the efficiency of biochemical reactions and biological
energy transport. It is thus crucial to uncover the mystery of the
hydration structure and dynamics as well as their coupling to collective
motions of proteins in aqueous solutions. Here, we report dielectric
properties of aqueous bovine serum albumin protein solutions as a
model system using an extremely sensitive dielectric spectrometer
with frequencies spanning from megahertz to terahertz. The dielectric
relaxation spectra reveal several polarization mechanisms at the molecular
level with different time constants and dielectric strengths, reflecting
the complexity of protein–water interactions. Combining the
effective-medium approximation and molecular dynamics simulations,
we have determined collective vibrational modes at terahertz frequencies
and the number of water molecules in the tightly bound and loosely
bound hydration layers. High-precision measurements of the number
of hydration water molecules indicate that the dynamical influence
of proteins extends beyond the first solvation layer, to around 7
Ă… distance from the protein surface, with the largest slowdown
arising from water molecules directly hydrogen-bonded to the protein.
Our results reveal critical information of protein dynamics and protein–water
interfaces, which determine biochemical functions and reactivity of
proteins
Fluorescence from Multiple Chromophore Hydrogen-Bonding States in the Far-Red Protein TagRFP675
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
Fluorescence from Multiple Chromophore Hydrogen-Bonding States in the Far-Red Protein TagRFP675
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
Fluorescence from Multiple Chromophore Hydrogen-Bonding States in the Far-Red Protein TagRFP675
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
Fluorescence from Multiple Chromophore Hydrogen-Bonding States in the Far-Red Protein TagRFP675
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