6 research outputs found
Excited State Structure and Dynamics of the Neutral and Anionic Flavin Radical Revealed by Ultrafast Transient Mid-IR to Visible Spectroscopy
Neutral and anionic flavin radicals are involved in numerous
photochemical
processes and play an essential part in forming the signaling state
of various photoactive flavoproteins such as cryptochromes and BLUF
domain proteins. A stable neutral radical flavin has been prepared
for study in aqueous solution, and both neutral and anion radical
states have been stabilized in the proteins flavodoxin and glucose
oxidase. Ultrafast transient absorption measurements were performed
in the visible and mid-infrared region in order to characterize the
excited state dynamics and the excited and ground state vibrational
spectra and to probe the effect of the protein matrix on them. These
data are compared with the results of density functional theory calculations.
Excited state decay dynamics were found to be a strong function of
the protein matrix. The ultrafast electron transfer quenching mechanism
of the excited flavin moiety in glucose oxidase is characterized by
vibrational spectroscopy. Such data will be critical in the ongoing
analysis of the photocycle of photoactive flavoproteins
Proteins in Action: Femtosecond to Millisecond Structural Dynamics of a Photoactive Flavoprotein
Living
systems are fundamentally dependent on the ability of proteins
to respond to external stimuli. The mechanism, the underlying structural
dynamics, and the time scales for regulation of this response are
central questions in biochemistry. Here we probe the structural dynamics
of the BLUF domain found in several photoactive flavoproteins, which
is responsible for light activated functions as diverse as phototaxis
and gene regulation. Measurements have been made over 10 decades of
time (from 100 fs to 1 ms) using transient vibrational spectroscopy.
Chromophore (flavin ring) localized dynamics occur on the pico- to
nanosecond time scale, while subsequent protein structural reorganization
is observed over microseconds. Multiple time scales are observed for
the dynamics associated with different vibrations of the protein,
suggesting an underlying hierarchical relaxation pathway. Structural
evolution in residues directly H-bonded to the chromophore takes place
more slowly than changes in more remote residues. However, a point
mutation which suppresses biological function is shown to ‘short
circuit’ this structural relaxation pathway, suppressing the
changes which occur further away from the chromophore while accelerating
dynamics close to it
Vibrational Assignment of the Ultrafast Infrared Spectrum of the Photoactivatable Flavoprotein AppA
The blue light using flavin (BLUF) domain proteins, such
as the transcriptional antirepressor AppA, are a novel class of photosensors
that bind flavin noncovalently in order to sense and respond to high-intensity
blue (450 nm) light. Importantly, the noncovalently bound flavin chromophore
is unable to undergo large-scale structural change upon light absorption,
and thus there is significant interest in understanding how the BLUF
protein matrix senses and responds to flavin photoexcitation. Light
absorption is proposed to result in alterations in the hydrogen-bonding
network that surrounds the flavin chromophore on an ultrafast time
scale, and the structural changes caused by photoexcitation are being
probed by vibrational spectroscopy. Here we report ultrafast time-resolved
infrared spectra of the AppA BLUF domain (AppA<sub>BLUF</sub>) reconstituted
with isotopes of FAD, specifically [U-<sup>13</sup>C<sub>17</sub>]-FAD,
[xylene-<sup>13</sup>C<sub>8</sub>]-FAD, [U-<sup>15</sup>N<sub>4</sub>]-FAD, and [4-<sup>18</sup>O<sub>1</sub>]-FAD both in solution and
bound to AppA<sub>BLUF</sub>. This allows for unambiguous assignment
of ground- and excited-state modes arising directly from the flavin.
Studies of model compounds and DFT calculations of the ground-state
vibrational spectra reveal the sensitivity of these modes to their
environment, indicating they can be used as probes of structural dynamics
Femtosecond to Millisecond Dynamics of Light Induced Allostery in the <i>Avena sativa</i> LOV Domain
The
rational engineering of photosensor proteins underpins the
field of optogenetics, in which light is used for spatiotemporal control
of cell signaling. Optogenetic elements function by converting electronic
excitation of an embedded chromophore into structural changes on the
microseconds to seconds time scale, which then modulate the activity
of output domains responsible for biological signaling. Using time-resolved
vibrational spectroscopy coupled with isotope labeling, we have mapped
the structural evolution of the LOV2 domain of the flavin binding
phototropin <i>Avena sativa</i> (AsLOV2) over 10 decades
of time, reporting structural dynamics between 100 fs and 1 ms after
optical excitation. The transient vibrational spectra contain contributions
from both the flavin chromophore and the surrounding protein matrix.
These contributions are resolved and assigned through the study of
four different isotopically labeled samples. High signal-to-noise
data permit the detailed analysis of kinetics associated with the
light activated structural evolution. A pathway for the photocycle
consistent with the data is proposed. The earliest events occur in
the flavin binding pocket, where a subpicosecond perturbation of the
protein matrix occurs. In this perturbed environment, the previously
characterized reaction between triplet state isoalloxazine and an
adjacent cysteine leads to formation of the adduct state; this step
is shown to exhibit dispersive kinetics. This reaction promotes coupling
of the optical excitation to successive time-dependent structural
changes, initially in the β-sheet and then α-helix regions
of the AsLOV2 domain, which ultimately gives rise to Jα-helix
unfolding, yielding the signaling state. This model is tested through
point mutagenesis, elucidating in particular the key mediating role
played by Q513
Elucidating the Signal Transduction Mechanism of the Blue-Light-Regulated Photoreceptor YtvA: From Photoactivation to Downstream Regulation
The blue-light photoreceptor
YtvA from Bacillus
subtilis has an N-terminal flavin mononucleotide (FMN)-binding
light-oxygen-voltage (LOV) domain that is fused to a C-terminal sulfate
transporter and anti-σ factor antagonist (STAS) output domain.
To interrogate the signal transduction pathway that leads to photoactivation,
the STAS domain was replaced with a histidine kinase, so that photoexcitation
of the flavin could be directly correlated with biological activity.
N94, a conserved Asn that is hydrogen bonded to the FMN C2O
group, was replaced with Ala, Asp, and Ser residues to explore the
role of this residue in triggering the structural dynamics that activate
the output domain. Femtosecond to millisecond time-resolved multiple
probe spectroscopy coupled with a fluorescence polarization assay
revealed that the loss of the hydrogen bond between N94 and the C2O
group decoupled changes in the protein structure from photoexcitation.
In addition, alterations in N94 also decreased the stability of the
Cys-FMN adduct formed in the light-activated state by up to a factor
of ∼25. Collectively, these studies shed light on the role
of the hydrogen bonding network in the LOV β-scaffold in signal
transduction
Mechanism of the AppA<sub>BLUF</sub> Photocycle Probed by Site-Specific Incorporation of Fluorotyrosine Residues: Effect of the Y21 p<i>K</i><sub>a</sub> on the Forward and Reverse Ground-State Reactions
The
transcriptional antirepressor AppA is a blue light using flavin
(BLUF) photoreceptor that releases the transcriptional repressor PpsR
upon photoexcitation. Light activation of AppA involves changes in
a hydrogen-bonding network that surrounds the flavin chromophore on
the nanosecond time scale, while the dark state of AppA is then recovered
in a light-independent reaction with a dramatically longer half-life
of 15 min. Residue Y21, a component of the hydrogen-bonding network,
is known to be essential for photoactivity. Here, we directly explore
the effect of the Y21 p<i>K</i><sub>a</sub> on dark state
recovery by replacing Y21 with fluorotyrosine analogues that increase
the acidity of Y21 by 3.5 pH units. Ultrafast transient infrared measurements
confirm that the structure of AppA is unperturbed by fluorotyrosine
substitution, and that there is a small (3-fold) change in the photokinetics
of the forward reaction over the fluorotyrosine series. However, reduction
of 3.5 pH units in the p<i>K</i><sub>a</sub> of Y21 increases
the rate of dark state recovery by 4000-fold with a Brønsted
coefficient of ∼1, indicating that the Y21 proton is completely
transferred in the transition state leading from light to dark adapted
AppA. A large solvent isotope effect of ∼6–8 is also
observed on the rate of dark state recovery. These data establish
that the acidity of Y21 is a crucial factor for stabilizing the light
activated form of the protein, and have been used to propose a model
for dark state recovery that will ultimately prove useful for tuning
the properties of BLUF photosensors for optogenetic applications