Blue Light Using Flavin Domain Research Articles

Origin of the multi-phasic quenching dynamics in the BLUF domains across the species

Blue light using flavin (BLUF) photoreceptors respond to light via one of nature’s smallest photo-switching domains. Upon photo-activation, the flavin cofactor in the BLUF domain exhibits multi-phasic dynamics, quenched by a proton-coupled electron transfer reaction involving the conserved Tyr and Gln. The dynamic behavior varies drastically across different species, the origin of which remains controversial. Here, we incorporate site-specific fluorinated Trp into three BLUF proteins, i.e., AppA, OaPAC and SyPixD, and characterize the percentages for the Wout, WinNHin and WinNHout conformations using 19F nuclear magnetic resonance spectroscopy. Using femtosecond spectroscopy, we identify that one key WinNHin conformation can introduce a branching one-step proton transfer in AppA and a two-step proton transfer in OaPAC and SyPixD. Correlating the flavin quenching dynamics with the active-site structural heterogeneity, we conclude that the quenching rate is determined by the percentage of WinNHin, which encodes a Tyr-Gln configuration that is not conducive to proton transfer.

Difference in the Charge-Separation Energetics between Distinct Conformers in the PixD Photoreceptor.

Blue light using flavin (BLUF) domain proteins are photoreceptors in various organisms. The PixD BLUF domain can adopt two conformations, W91out and W91in, with Trp91 either proximal or distal to flavin (FMN). Using a quantum mechanical/molecular mechanical/polarizable continuum model approach, the energetics of charge-separated and biradical states in the two conformations were investigated. In the W91out conformation, the charge-separated state (FMN•-) is more stable than the photoexcited state (FMN*), whereas it is less stable due to an electrostatic repulsive interaction with the Ser28 side chain in the W91in conformation. This leads to a lower activation energy for the charge separation in the W91out conformation, resulting in a faster charge separation compared to that in the W91in conformation. In the W91out conformation, the radical state (FMNH•) is more stable than FMN•- and forms from FMN•-, leading to reorientation of the Gln50 side chain adjacent to FMN and formation of a hydrogen bond between Gln50 and FMN. Subsequently, a signaling state forms through charge recombination. In contrast, in the W91in conformation, FMN•- cannot proceed further, returning to the dark-adapted state, as FMNH• is less stable. Thus, formation of the signaling state exclusively occurs in the W91out conformation.

Single Amino Acid Mutation Decouples Photochemistry of the BLUF Domain from the Enzymatic Function of OaPAC and Drives the Enzyme to a Switched-on State

Photoactivated adenylate cyclases (PACs) are light-activated enzymes that combine a BLUF (blue-light using flavin) domain and an adenylate cyclase domain that are able to increase the levels of the important second messenger cAMP (cyclic adenosine monophosphate) upon blue-light excitation. The light-induced changes in the BLUF domain are transduced to the adenylate cyclase domain via a mechanism that has not yet been established. One critical residue in the photoactivation mechanism of BLUF domains, present in the vicinity of the flavin is the glutamine amino acid close to the N5 of the flavin. The role of this residue has been investigated extensively both experimentally and theoretically. However, its role in the activity of the photoactivated adenylate cyclase, OaPAC has never been addressed. In this work, we applied ultrafast transient visible and infrared spectroscopies to study the photochemistry of the Q48E OaPAC mutant. This mutation altered the primary electron transfer process and switched the enzyme into a permanent ‘on’ state, able to increase the cAMP levels under dark conditions compared to the cAMP levels of the dark-adapted state of the wild-type OaPAC. Differential scanning calorimetry measurements point to a less compact structure for the Q48E OaPAC mutant. The ensemble of these findings provide insight into the important elements in PACs and how their fine tuning may help in the design of optogenetic devices.

Integrated Computational Study of the Light-Activated Structure of the AppA BLUF Domain and Its Spectral Signatures.

We apply an integrated approach combining microsecond MD simulations and (polarizable) QM/MM calculations of NMR, FTIR, and UV-vis spectra to validate the structure of the light-activated form of the AppA photoreceptor, an example of blue light using flavin (BLUF) protein domain. The latter photoactivate through a proton-coupled electron transfer (PCET) that results in a tautomerization of a conserved glutamine residue in the active site, but this mechanism has never been spectroscopically proven for AppA, which has been always considered as an exception. Our simulations instead confirm that the spectral features observed upon AppA photoactivation are indeed directly connected to the tautomer form of glutamine as predicted by the PCET mechanism. In addition, we observe small but significant changes in the AppA structure, which are transmitted from the flavin binding pocket to the surface of the protein.

The nature of proton-coupled electron transfer in a blue light using flavin domain

Proton-coupled electron transfer (PCET) is key to the activation of the blue light using flavin (BLUF) domain photoreceptors. Here, to elucidate the photocycle of the central FMN-Gln-Tyr motif in the BLUF domain of OaPAC, we eliminated the intrinsic interfering W90 in the mutant design. We integrated the stretched exponential function into the target analysis to account for the dynamic heterogeneity arising from the active-site solvation relaxation and the flexible H-bonding network as shown in the molecular dynamics simulation results, facilitating a simplified expression of the kinetics model. We find that, in both the functional wild-type (WT) and the nonfunctional Q48E and Q48A, forward PCET happens in the range of 105 ps to 344 ps, with a kinetic isotope effect (KIE) measured to be ∼1.8 to 2.4, suggesting that the nature of the forward PCET is concerted. Remarkably, only WT proceeds with an ultrafast reverse PCET process (31 ps, KIE = 4.0), characterized by an inverted kinetics of the intermediate FMNH˙. Our results reveal that the reverse PCET is driven by proton transfer via an intervening imidic Gln.

Photophysics of the Blue Light Using Flavin Domain

Light activated proteins are at the heart of photobiology and optogenetics, so there is wide interest in understanding the mechanisms coupling optical excitation to protein function. In addition, such light activated proteins provide unique insights into the real-time dynamics of protein function. Using pump-probe spectroscopy, the function of a photoactive protein can be initiated by a sub-100 fs pulse of light, allowing subsequent protein dynamics to be probed from femtoseconds to milliseconds and beyond. Among the most interesting photoactive proteins are the blue light using flavin (BLUF) domain proteins, which regulate the response to light of a wide range of bacterial and some euglenoid processes. The photosensing mechanism of BLUF domains has long been a subject of debate. In contrast to other photoactive proteins, the electronic and nuclear structure of the chromophore (flavin) is the same in dark- and light-adapted states. Thus, the driving force for photoactivity is unclear.To address this question requires real-time observation of both chromophore excited state processes and their effect on the structure and dynamics of the surrounding protein matrix. In this Account we describe how time-resolved infrared (IR) experiments, coupled with chemical biology, provide important new insights into the signaling mechanism of BLUF domains. IR measurements are sensitive to changes in both chromophore electronic structure and protein hydrogen bonding interactions. These contributions are resolved by isotope labeling of the chromophore and protein separately. Further, a degree of control over BLUF photochemistry is achieved through mutagenesis, while unnatural amino acid substitution allows us to both fine-tune the photochemistry and time resolve protein dynamics with spatial resolution.Ultrafast studies of BLUF domains reveal non-single-exponential relaxation of the flavin excited state. That relaxation leads within one nanosecond to the original flavin ground state bound in a modified hydrogen-bonding network, as seen in transient and steady-state IR spectroscopy. The change in H-bond configuration arises from formation of an unusual enol (imine) form of a critical glutamine residue. The dynamics observed, complemented by quantum mechanical calculations, suggest a unique sequential electron then double proton transfer reaction as the driving force, followed by rapid reorganization in the binding site and charge recombination. Importantly, studies of several BLUF domains reveal an unexpected diversity in their dynamics, although the underlying structure appears highly conserved. It is suggested that this diversity reflects structural dynamics in the ground state at standard temperature, leading to a distribution of structures and photochemical outcomes. Time resolved IR measurements were extended to the millisecond regime for one BLUF domain, revealing signaling state formation on the microsecond time scale. The mechanism involves reorganization of a β-sheet connected to the chromophore binding pocket via a tryptophan residue. The potential of site-specific labeling amino acids with IR labels as a tool for probing protein structural dynamics was demonstrated.In summary, time-resolved IR studies of BLUF domains (along with related studies at visible wavelengths and quantum and molecular dynamics calculations) have resolved the photoactivation mechanism and real-time dynamics of signaling state formation. These measurements provide new insights into protein structural dynamics and will be important in optimizing the potential of BLUF domains in optobiology.

From crystallographic data to the solution structure of photoreceptors: the case of the AppA BLUF domain.

Photoreceptor proteins bind a chromophore, which, upon light absorption, modifies its geometry or its interactions with the protein, finally inducing the structural change needed to switch the protein from an inactive to an active or signaling state. In the Blue Light-Using Flavin (BLUF) family of photoreceptors, the chromophore is a flavin and the changes have been connected with a rearrangement of the hydrogen bond network around it on the basis of spectroscopic changes measured for the dark-to-light conversion. However, the exact conformational change triggered by the photoexcitation is still elusive mainly because a clear consensus on the identity not only of the light activated state but also of the dark one has not been achieved. Here, we present an integrated investigation that combines microsecond MD simulations starting from the two conflicting crystal structures available for the AppA BLUF domain with calculations of NMR, IR and UV-Vis spectra using a polarizable QM/MM approach. Thanks to such a combined analysis of the three different spectroscopic responses, a robust characterization of the structure of the dark state in solution is given together with the uncovering of important flaws of the most popular molecular mechanisms present in the literature for the dark-to-light activation.

Formation of an unusual glutamine tautomer in a blue light using flavin photocycle characterizes the light-adapted state

Blue light using flavin (BLUF) photoreceptor proteins are critical for many light-activated biological processes and are promising candidates for optogenetics because of their modular nature and long-range signaling capabilities. Although the photocycle of the Slr1694 BLUF domain has been characterized experimentally, the identity of the light-adapted state following photoexcitation of the bound flavin remains elusive. Herein hybrid quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations of this photocycle provide a nonequilibrium dynamical picture of a possible mechanism for the formation of the light-adapted state. Photoexcitation of the flavin induces a forward proton-coupled electron transfer (PCET) process that leads to the formation of an imidic acid tautomer of Gln50. The calculations herein show that the subsequent rotation of Gln50 allows a reverse PCET process that retains this tautomeric form. In the resulting purported light-adapted state, the glutamine tautomer forms a hydrogen bond with the flavin carbonyl group. Additional ensemble-averaged QM/MM calculations of the dark-adapted and purported light-adapted states demonstrate that the light-adapted state with the imidic acid glutamine tautomer reproduces the experimentally observed spectroscopic signatures. Specifically, the calculations reproduce the red shifts in the flavin electronic absorption and carbonyl stretch infrared spectra in the light-adapted state. Further hydrogen-bonding analyses suggest the formation of hydrogen-bonding interactions between the flavin and Arg65 in the light-adapted state, providing a plausible explanation for the experimental observation of faster photoinduced PCET in this state. These characteristics of the light-adapted state may also be essential for the long-range signaling capabilities of this photoreceptor protein.

Functional dynamics of a single tryptophan residue in a BLUF protein revealed by fluorescence spectroscopy

Blue Light Using Flavin (BLUF) domains are increasingly being adopted for use in optogenetic constructs. Despite this, much remains to be resolved on the mechanism of their activation. The advent of unnatural amino acid mutagenesis opens up a new toolbox for the study of protein structural dynamics. The tryptophan analogue, 7-aza-Trp (7AW) was incorporated in the BLUF domain of the Activation of Photopigment and pucA (AppA) photoreceptor in order to investigate the functional dynamics of the crucial W104 residue during photoactivation of the protein. The 7-aza modification to Trp makes selective excitation possible using 310 nm excitation and 380 nm emission, separating the signals of interest from other Trp and Tyr residues. We used Förster energy transfer (FRET) between 7AW and the flavin to estimate the distance between Trp and flavin in both the light- and dark-adapted states in solution. Nanosecond fluorescence anisotropy decay and picosecond fluorescence lifetime measurements for the flavin revealed a rather dynamic picture for the tryptophan residue. In the dark-adapted state, the major population of W104 is pointing away from the flavin and can move freely, in contrast to previous results reported in the literature. Upon blue-light excitation, the dominant tryptophan population is reorganized, moves closer to the flavin occupying a rigidly bound state participating in the hydrogen-bond network around the flavin molecule.

Site-Specific Protein Dynamics Probed by Ultrafast Infrared Spectroscopy of a Noncanonical Amino Acid.

Real-time observation of structure changes associated with protein function remains a major challenge. Ultrafast pump-probe methods record dynamics in light activated proteins, but the assignment of spectroscopic observables to specific structure changes can be difficult. The BLUF (blue light using flavin) domain proteins are an important class of light sensing flavoprotein. Here, we incorporate the unnatural amino acid (UAA) azidophenylalanine (AzPhe) at key positions in the H-bonding environment of the isoalloxazine chromophore of two BLUF domains, namely, PixD and AppABLUF; both proteins retain the red-shift on irradiation characteristic of photoactivity. Steady state and ultrafast time resolved infrared difference measurements of the azido mode reveal site-specific information on the nature and dynamics of light driven structure change. AzPhe dynamics are thus shown to be an effective probe of BLUF domain photoactivation, revealing significant differences between the two proteins and a differential response of the two sites to chromophore excitation.

Modular Diversity of the BLUF Proteins and Their Potential for the Development of Diverse Optogenetic Tools

Organisms can respond to varying light conditions using a wide range of sensory photoreceptors. These photoreceptors can be standalone proteins or represent a module in multidomain proteins, where one or more modules sense light as an input signal which is converted into an output response via structural rearrangements in these receptors. The output signals are utilized downstream by effector proteins or multiprotein clusters to modulate their activity, which could further affect specific interactions, gene regulation or enzymatic catalysis. The blue-light using flavin (BLUF) photosensory module is an autonomous unit that is naturally distributed among functionally distinct proteins. In this study, we identified 34 BLUF photoreceptors of prokaryotic and eukaryotic origin from available bioinformatics sequence databases. Interestingly, our analysis shows diverse BLUF-effector arrangements with a functional association that was previously unknown or thought to be rare among the BLUF class of sensory proteins, such as endonucleases, tet repressor family (tetR), regulators of G-protein signaling, GAL4 transcription family and several other previously unidentified effectors, such as RhoGEF, Phosphatidyl-Ethanolamine Binding protein (PBP), ankyrin and leucine-rich repeats. Interaction studies and the indexing of BLUF domains further show the diversity of BLUF-effector combinations. These diverse modular architectures highlight how the organism’s behaviour, cellular processes, and distinct cellular outputs are regulated by integrating BLUF sensing modules in combination with a plethora of diverse signatures. Our analysis highlights the modular diversity of BLUF containing proteins and opens the possibility of creating a rational design of novel functional chimeras using a BLUF architecture with relevant cellular effectors. Thus, the BLUF domain could be a potential candidate for the development of powerful novel optogenetic tools for its application in modulating diverse cell signaling.

Propensity for Proton Relay and Electrostatic Impact of Protein Reorganization in Slr1694 BLUF Photoreceptor.

Photoreceptor proteins play a vital role in a wide range of light-regulated processes. The formation of the light-adapted state of blue light using flavin (BLUF) photoreceptors is thought to involve rearrangements of hydrogen-bonding networks upon photoexcitation. Free energy simulations with partial charges corresponding to relevant ground and excited states of the Slr1694 BLUF domain characterize conformations prior to and following photoexcitation. The simulations indicate that Trp91 is thermodynamically favored to be in the active site, although it is also able to sample conformations outside the active site. For experimentally observed conformations of Trp91, Gln50 is thermodynamically favored to be oriented for a proton relay bridging Tyr8 and the flavin. When Trp91 is rotated such that it can donate a hydrogen bond to Gln50, as observed in other BLUF domains, the proton relay is not thermodynamically favored in the ground state, providing a possible explanation for the relatively fast photocycle of the Slr1694 BLUF domain. Photoexcitation to the locally excited (LE) state of the flavin induces the formation of the proton relay if it is not already formed. Electrostatically embedded time-dependent density functional theory calculations indicate that the proton relay reduces the energy gap between the LE state and the charge-transfer (CT) state associated with electron transfer from Tyr8 to the flavin. Although the CT state is higher in energy than the LE state prior to photoexcitation, the protein environment can reorganize in a manner that stabilizes the CT state so that it is lower than the LE state, enabling the LE to CT state transition. An electrostatic analysis identifies motions of individual residues, such as Arg65, that stabilize electron transfer from Tyr8 to the flavin. These conformational changes facilitate the critical proton-coupled electron transfer reaction in the BLUF photocycle.

Structural dynamics of blue light sensitive bacterial photoreceptor blsA

Blue light sensing A (blsA) is a blue light using flavin (BLUF) domain isolated from opportunistic pathogen Acinetobacter baumanii. BlsA is vital for A. baumanii’s motility, biofilm formation and virulence. When blue light activates blsA [in the N‐terminus], the protein interacts with its effector domain(s) [in the C‐terminus] with implications in downstream signaling events. Photo absorption modifies the hydrogen bonds and the electrostatic interactions between flavin and the BLUF domain with consequent structural changes that are integral in the signal transduction. Spectroscopic data highlight involvement of residues glutamine (51) and tryptophan (92) in the photoactivated signaling state of blsA. My goal is to solve blsA's structure using x‐ray crystallography to determine the specific residues involved in the signaling state. Comparison of crystal structures of blsA (~1.8 Å) in the dark state and the photoactivated light state does not show movement of the residues glutamine and tryptophan in the two states. However, we detect a distinct conformational change of an alpha‐helix in the C‐terminus‐ the effector protein binding terminus‐ of blsA.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Seeing the light with BLUF proteins.

First described about 15years ago, BLUF (Blue Light Using Flavin) domains are light-triggered switches that control enzyme activity or gene expression in response to blue light, remaining activated for seconds or even minutes after stimulation. The conserved, ferredoxin-like fold holds a flavin chromophore that captures the light and somehow triggers downstream events. BLUF proteins are found in both prokaryotes and eukaryotes and have a variety of architectures and oligomeric forms, but the BLUF domain itself seems to have a well-preserved structure and mechanism that have been the focus of intense study for a number of years. Crystallographic and NMR structures of BLUF domains have been solved, but the conflicting models have led to considerable debate about the atomic details of photo-activation. Advanced spectroscopic and computational methods have been used to analyse the early events after photon absorption, but these too have led to widely differing conclusions. New structural models are improving our understanding of the details of the mechanism and may lead to novel tailor-made tools for optogenetics.

Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium

Cyclic-AMP is one of the most important second messengers, regulating many crucial cellular events in both prokaryotes and eukaryotes, and precise spatial and temporal control of cAMP levels by light shows great promise as a simple means of manipulating and studying numerous cell pathways and processes. The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) is a small homodimer eminently suitable for this task, requiring only a simple flavin chromophore within a blue light using flavin (BLUF) domain. These domains, one of the most studied types of biological photoreceptor, respond to blue light and either regulate the activity of an attached enzyme domain or change its affinity for a repressor protein. BLUF domains were discovered through studies of photo-induced movements of Euglena gracilis, a unicellular flagellate, and gene expression in the purple bacterium Rhodobacter sphaeroides, but the precise details of light activation remain unknown. Here, we describe crystal structures and the light regulation mechanism of the previously undescribed OaPAC, showing a central coiled coil transmits changes from the light-sensing domains to the active sites with minimal structural rearrangement. Site-directed mutants show residues essential for signal transduction over 45 Å across the protein. The use of the protein in living human cells is demonstrated with cAMP-dependent luciferase, showing a rapid and stable response to light over many hours and activation cycles. The structures determined in this study will assist future efforts to create artificial light-regulated control modules as part of a general optogenetic toolkit.

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.

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.

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.

A PixD—PapB Chimeric Protein Reveals the Function of the BLUF Domain C-Terminal α-Helices for Light Signal Transduction

Blue light-using flavin (BLUF) proteins form a subfamily of blue light photoreceptors, are found in many bacteria and algae, and are further classified according to their structures. For one type of BLUF-containing protein, e.g. PixD, the central axes of its two C-terminal α-helices are perpendicular to the β-sheet of its N-terminal BLUF domain. For another type, e.g. PapB, the central axes of its two C-terminal α-helices are parallel to its BLUF domain β-sheet. However, the functional significance of the different orientations with respect to phototransduction is not clear. For the study reported herein, we constructed a chimeric protein, Pix0522, containing the core of the PixD BLUF domain and the C-terminal region of PapB, including the two α-helices, and characterized its biochemical and spectroscopic properties. Fourier transform infrared spectroscopy detected similar light-induced conformational changes in the C-terminal α-helices of Pix0522 and PapB. Pix0522 interacts with and activates the PapB-interacting enzyme, PapA, demonstrating the functionality of Pix0522. These results provide direct evidence that the BLUF C-terminal α-helices function as an intermediary that accepts the flavin-sensed blue light signal and transmits it downstream during phototransduction.

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.