Mechanism of the AppABLUF Photocycle Probed by Site-Specific Incorporation of Fluorotyrosine Residues: Effect of the Y21 pKa 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 pKa 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 pKa 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.

Dissecting the Molecular Roles of Histone Chaperones in Histone Acetylation by Type B Histone Acetyltransferases (HAT-B).

The HAT-B enzyme complex is responsible for acetylating newly synthesized histone H4 on lysines K5 and K12. HAT-B is a multisubunit complex composed of the histone acetyltransferase 1 (Hat1) catalytic subunit and the Hat2 (rbap46) histone chaperone. Hat1 is predominantly localized in the nucleus as a member of a trimeric NuB4 complex containing Hat1, Hat2, and a histone H3-H4 specific histone chaperone called Hif1 (NASP). In addition to Hif1 and Hat2, Hat1 interacts with Asf1 (anti-silencing function 1), a histone chaperone that has been reported to be involved in both replication-dependent and -independent chromatin assembly. To elucidate the molecular roles of the Hif1 and Asf1 histone chaperones in HAT-B histone binding and acetyltransferase activity, we have characterized the stoichiometry and binding mode of Hif1 and Asf1 to HAT-B and the effect of this binding on the enzymatic activity of HAT-B. We find that Hif1 and Asf1 bind through different modes and independently to HAT-B, whereby Hif1 binds directly to Hat2, and Asf1 is only capable of interactions with HAT-B through contacts with histones H3-H4. We also demonstrate that HAT-B is significantly more active against an intact H3-H4 heterodimer over a histone H4 peptide, independent of either Hif1 or Asf1 binding. Mutational studies further demonstrate that HAT-B binding to the histone tail regions is not sufficient for this enhanced activity. Based on these data, we propose a model for HAT-B/histone chaperone assembly and acetylation of H3-H4 complexes.

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.

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 ß5 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.

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.

Protein photochromism observed by ultrafast vibrational spectroscopy.

Photochromic proteins, such as Dronpa, are of particular importance in bioimaging and form the basis of ultraresolution fluorescence microscopy. The photochromic reaction involves switching between a weakly emissive neutral trans form of the chromophore (A) and its emissive cis anion (B). Controlling the rates of switching has the potential to significantly enhance the spatial and temporal resolution in microscopy. However, the mechanism of the switching reaction has yet to be established. Here we report a high signal-to-noise ultrafast transient infrared investigation of the photochromic reaction in the mutant Dronpa2, which exhibits facile switching behavior. In these measurements we excite both the A and B forms and observe the evolution in the IR difference spectra over hundreds of picoseconds. Electronic excitation leads to bleaching of the ground electronic state and instantaneous (subpicosecond) changes in the vibrational spectrum of the protein. The chromophore and protein modes evolve with different kinetics. The chromophore ground state recovers in a fast nonsingle-exponential relaxation, while in a competing reaction the protein undergoes a structural change. This results in formation of a metastable form of the protein in its ground electronic state (A'), which subsequently evolves on the time scale of hundreds of picoseconds. The changes in the vibrational spectrum that occur on the subnanosecond time scale do not show unambiguous evidence for either proton transfer or isomerization, suggesting that these low-yield processes occur from the metastable state on a longer time scale and are thus not the primary photoreaction. Formation of A', and further relaxation of this state to the cis anion B, are relatively rare events, thus accounting for the overall low yield of the photochemical reaction.

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(BLUF)) reconstituted with isotopes of FAD, specifically [U-(13)C(17)]-FAD, [xylene-(13)C(8)]-FAD, [U-(15)N(4)]-FAD, and [4-(18)O(1)]-FAD both in solution and bound to AppA(BLUF). 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.

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.

Ultrafast transient mid IR to visible spectroscopy of fully reduced flavins.

The light sensing apparatus of many organisms includes a flavoprotein. In any spectroscopic analysis of the photocycle of flavoproteins a detailed knowledge of the spectroscopy and excited state dynamics of potential intermediates is required. Here we correlate transient vibrational and electronic spectra of the two fully reduced forms of flavin adenine dinucleotide (FAD): FADH(-) and FADH(2). Ground and excited state frequencies of the characteristic carbonyl modes are observed and assigned with the aid of DFT calculations. Excited state decay and ground state recovery dynamics of the two states are reported. Excited state decay occurs on the picosecond timescale, in agreement with the low fluorescence yield, and is markedly non single exponential in FADH(-). Further, an unusual 'inverse' isotope effect is observed in the decay time of FADH(-), suggesting the involvement in the radiationless relaxation coordinate of an NH or hydrogen bond mode that strengthens in the excited electronic state. Ground state recovery also occurs on the picosecond time scale, consistent with radiationless decay by internal conversion, but is slower than the excited state decay.

Photoexcitation of the blue light using FAD photoreceptor AppA results in ultrafast changes to the protein matrix.

Photoexcitation of the flavin chromophore in the BLUF photosensor AppA results in a conformational change that leads to photosensor activation. This conformational change is mediated by a hydrogen-bonding network that surrounds the flavin, and photoexcitation is known to result in changes in the network that include a strengthening of hydrogen bonding to the flavin C4═O carbonyl group. Q63 is a key residue in the hydrogen-bonding network, and replacement of this residue with a glutamate results in a photoinactive mutant. While the ultrafast time-resolved infrared (TRIR) spectrum of Q63E AppA(BLUF) is characterized by flavin carbonyl modes at 1680 and 1650 cm(-1), which are similar in frequency to the analogous modes from the light activated state of the wild-type protein, a band is also observed in the TRIR spectrum at 1724 cm(-1) that is unambiguously assigned to the Q63E carboxylic acid based on U-(13)C labeling of the protein. Light absorption instantaneously (<100 fs) bleaches the 1724 cm(-1) band leading to a transient absorption at 1707 cm(-1). Because Q63E is not part of the isoalloxazine electronic transition, the shift in frequency must arise from a sub picosecond perturbation to the flavin binding pocket. The light-induced change in the frequency of the Q63E side chain is assigned to an increase in hydrogen-bond strength of 3 kcal mol(-1) caused by electronic reorganization of the isoalloxazine ring in the excited state, providing direct evidence that the protein matrix of AppA responds instantaneously to changes in the electronic structure of the chromophore and supporting a model for photoactivation of the wild-type protein that involves initial tautomerization of the Q63 side chain.

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 (AppA(BLUF)) 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.