17 research outputs found
Ultrafast Structural Dynamics of BlsA, a Photoreceptor from the Pathogenic Bacterium Acinetobacter baumannii
Acinetobacter baumannii is an important human pathogen that can form biofilms and persist under harsh environmental conditions. Biofilm formation and virulence are modulated by blue light, which is thought to be regulated by a BLUF protein, BlsA. To understand the molecular mechanism of light sensing, we have used steady-state and ultrafast vibrational spectroscopy to compare the photoactivation mechanism of BlsA to the BLUF photosensor AppA from Rhodobacter sphaeroides. Although similar photocycles are observed, vibrational data together with homology modeling identify significant differences in the β5 strand in BlsA caused by photoactivation, which are proposed to be directly linked to downstream signaling
BLUF Domain Function Does Not Require a Metastable Radical Intermediate State
BLUF
(blue light using flavin) domain proteins are an important
family of blue light-sensing proteins which control a wide variety
of functions in cells. The primary light-activated step in the BLUF
domain is not yet established. A number of experimental and theoretical
studies points to a role for photoinduced electron transfer (PET)
between a highly conserved tyrosine and the flavin chromophore to
form a radical intermediate state. Here we investigate the role of
PET in three different BLUF proteins, using ultrafast broadband transient
infrared spectroscopy. We characterize and identify infrared active
marker modes for excited and ground state species and use them to
record photochemical dynamics in the proteins. We also generate mutants
which unambiguously show PET and, through isotope labeling of the
protein and the chromophore, are able to assign modes characteristic
of both flavin and protein radical states. We find that these radical
intermediates are not observed in two of the three BLUF domains studied,
casting doubt on the importance of the formation of a population of
radical intermediates in the BLUF photocycle. Further, unnatural amino
acid mutagenesis is used to replace the conserved tyrosine with fluorotyrosines,
thus modifying the driving force for the proposed electron transfer
reaction; the rate changes observed are also not consistent with a
PET mechanism. Thus, while intermediates of PET reactions can be observed
in BLUF proteins they are not correlated with photoactivity, suggesting
that radical intermediates are not central to their operation. Alternative
nonradical pathways including a keto–enol tautomerization induced
by electronic excitation of the flavin ring are considered
Electron Transfer Quenching in Light Adapted and Mutant Forms of the AppA BLUF Domain
The Blue Light Using Flavin (BLUF) domain proteins are an important family of photoreceptors controlling a range of responses in a wide variety of organisms. The details of the primary photochemical mechanism, by which light absorption in the isoalloxazine ring of the flavin is converted into a structure change to form the signalling state of the protein, is unresolved. In this work we apply ultrafast time resolved infra-red (TRIR) spectroscopy to investigate the primary photophysics of the BLUF domain of the protein AppA (AppABLUF) a light activated antirepressor. Here a number of mutations at Y21 and W104 in AppABLUF are investigated. The Y21 mutants are known to be photoinactive, while W104 mutants show the characteristic spectral red-shift associated with BLUF domain activity. Using TRIR we observed separately the decay of the excited state and the recovery of the ground state. In both cases the kinetics are found to be non-single exponential for all the proteins studied, suggesting a range of ground state structures. In the Y21 mutants an intermediate state was also observed, assigned to formation of the radical of the isoalloxazine (flavin) ring. The electron donor is the W104 residue. In contrast, no radical intermediates were detected in the studies of the photoactive dark adapted proteins, dAppABLUF and the dW104 mutants, suggesting a structure change in the Y21 mutants which favours W104 to isoalloxazine electron transfer. In contrast, in the light adapted form of the proteins (lAppABLUF, lW104) a radical intermediate was detected and the kinetics were greatly accelerated. In this case the electron donor was Y21 and major structural changes are associated with the enhanced quenching. In AppABLUF and the seven mutants studied radical intermediates are readily observed by TRIR spectroscopy, but there is no correlation with photoactivity. This suggests that if a charge separated state has a role in the BLUF photocycle it is only as a very short lived intermediate.</p
Ultrafast Infrared Spectroscopy of an Isotope-Labeled Photoactivatable Flavoprotein
The blue light using flavin (BLUF) domain photosensors, such as the transcriptional antirepressor AppA, utilize a noncovalently bound flavin as the chromophore for photoreception. Since the isoalloxazine ring of the chromophore is unable to undergo large-scale structural change upon light absorption, there is intense 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 (AppABLUF) reconstituted with isotopically labeled riboflavin (Rf) and flavin adenine dinucleotide (FAD), which permit the first unambiguous assignment of ground and excited state modes arising directly from the flavin carbonyl groups. Studies of model compounds and DFT calculations of the ground state vibrational spectra reveal the sensitivity of these modes to their environment, indicating that they can be used as probes of structural dynamics
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