Light-induced flipping of a conserved glutamine sidechain and its orientation in the AppA BLUF domain

The AppA BLUF domain is a blue light photoreceptor containing flavin. Conserved glutamine 63 is necessary for the photocycle of the protein, and its side chain has been proposed to flip in response to blue light illumination. Recently published crystal structures of AppA WT and the AppA mutant C20S describe contradictory conclusions regarding the orientation of the conserved glutamine 63 side chain in the dark. Here, we present evidence from NMR spectroscopy confirming light-induced flipping of the glutamine side chain to form a strong hydrogen bond between the glutamine 63 side chain carbonyl group and the tyrosine 21 side chain hydroxyl proton in the light-induced state. Our conclusions are consistent with published data from UV/vis absorbance and FTIR spectroscopy, as well as the crystal structure of AppA WT.     

Orientation of a key glutamine residue in the BLUF domain from AppA revealed by mutagenesis, spectroscopy, and quantum chemical calculations

The flavin-adenine-dinucleotide-binding BLUF domain constitutes a new class of blue-light receptors, and the N-terminal domain of AppA is a representative of this family. A crystal structure of the BLUF domain from AppA suggested that a conserved Gln63 forms a hydrogen bond with the flavin N5 atom. Upon light excitation, this residue is proposed to undergo a approximately 180 degrees rotation that leads to a rearrangement of a hydrogen bonding network. However, crystallographic studies on the other BLUF proteins claimed an opposite orientation for the glutamine residue. In this communication, we have revealed the presence of a Gln63-to-N5 hydrogen bond in the dark state of AppA by a combined approach of mutagenesis, spectroscopy, and quantum chemical calculations. The present finding supports the view that the reorientation of the Gln63 side chain is a key event in the signaling state formation of BLUF proteins.     

Computational characterization of reaction intermediates in the photocycle of the sensory domain of the AppA blue light photoreceptor

The AppA protein with the BLUF (blue light using flavin adenine dinucleotide) domain is a blue light photoreceptor that cycle between dark-adapted and light-induced functional states. We characterized possible reaction intermediates in the photocycle of AppA BLUF. Molecular dynamics (MD), quantum chemical and quantum mechanical-molecular mechanical (QM/MM) calculations were carried out to describe several stable structures of a molecular system modeling the protein. The coordinates of heavy atoms from the crystal structure (PDB code 2IYG) of the protein in the dark state served as starting point for 10 ns MD simulations. Representative MD frames were used in QM(B3LYP/cc-pVDZ)/MM(AMBER) calculations to locate minimum energy configurations of the model system. Vertical electronic excitation energies were estimated for the molecular clusters comprising the quantum subsystems of the QM/MM optimized structures using the SOS-CIS(D) quantum chemistry method. Computational results support the occurrence of photoreaction intermediates that are characterized by spectral absorption bands between those of the dark and light states. They agree with crystal structures of reaction intermediates (PDB code 2IYI) observed in the AppA BLUF domain. Transformations of the Gln63 side chain stimulated by photo-excitation and performed with the assistance of the chromophore and the Met106 side chain are responsible for these intermediates.     

The photoreaction mechanism in the bacterial blue light receptor BLUF according to metadynamics modeling

The mechanism of chemical transformations in the blue light photoreceptor domains (BLUF) implies the isomerization of the glutamine side chain. The Helmholtz energy profiles for the side-chain isomerization of the tautomeric form of glutamine in the BLUF domain of the bacterial protein AppA were calculated using metadynamics and the potentials that were obtained using quantum mechanics-molecular mechanics approximation (QM/MM).     

Theoretical study of proton coupled electron transfer reaction in the light state of the AppA BLUF photoreceptor

The BLUF (blue light sensor using flavin adenine dinucleotide) domain is widely studied as a prototype for proton coupled electron transfer (PCET) reactions in biological systems. In this work, the photo-induced concerted PCET reaction from the light state of the AppA BLUF domain is investigated. To model the simultaneous transfer of two protons in the reaction, two-dimensional potential energy surfaces for the double proton transfer are first calculated for the locally excited and charge transfer states, which are then used to obtain the vibrational wave function overlaps and the vibrational energy levels. Contributions to the PCET rate constant from each pair of vibronic states are then analyzed using the theory based on the Fermi's golden rule. We show that, the recently proposed light state structure of the BLUF domain with a tautomerized Gln63 residue is consistent with the concerted transfer of one electron and two protons. It is also found that, thermal fluctuations of the protein structure, especially the proton donor-acceptor distances, play an important role in determining the PCET reaction rate. © 2018 Wiley Periodicals, Inc.     

In Vivo Effects on Photosynthesis Gene Expression of Base Pair Exchanges in the Gene Encoding the Light-responsive BLUF Domain of AppA in Rhodobacter Sphaeroides

The Rhodobacter sphaeroides protein AppA has the unique quality of sensing and transmitting light and redox signals. By acting as antirepressor to the PpsR protein, it acts as a major regulator in photosynthesis gene expression. In this study, we show that by introducing amino acid exchanges into the AppA protein, the in vivo activity as an antirepressor can be greatly altered. The tryptophan 104 to phenylalanine (W104F) base exchange greatly diminished blue-light sensitivity of the BLUF domain. From the obtained in vivo data, the difference in thermal recovery rate of the signaling state of the BLUF domain between the wild type and mutated protein was calculated, predicting an about 10-fold faster recovery in the mutant, which is consistent with in vitro data. Introduction of a tyrosine 21 to phenylalanine (Y21F) or to cysteine (Y21C) mutation led to a complete loss of AppA antirepressor activity, while additionally leading to an increase of photosynthesis gene expression after illumination with high blue-light quantities. Interestingly, this effect is not visible in a W104F/Y21F double mutant that again shows a wild-type–like behavior of the BLUF domain after blue-light illumination, thus restoring the activity of AppA.     

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.     

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.

With an integrated molecular dynamics and QM/MM strategy we characterize the dark-state structure of a BLUF photoreceptor and ration alize the discrepancy between published crystal structures.     

Initial characterization of the primary photochemistry of AppA, a blue-light-using flavin adenine dinucleotide-domain containing transcriptional antirepressor protein from Rhodobacter sphaeroides: a key role for reversible intramolecular proton transfer from the flavin adenine dinucleotide chromophore to a conserved tyrosine?

The flavin adenine dinucleotide (FAD)-containing photoreceptor protein AppA (in which the FAD is bound to a novel so-called BLUF domain) from the purple nonsulfur bacterium Rhodobacter sphaeroides was previously shown to be photoactive by the formation of a slightly redshifted long-lived intermediate that is thought to be the signaling state. In this study, we provide further characterization of the primary photochemistry of this photoreceptor protein using UV-Vis and Fourier-transform infrared spectroscopy, pH measurements and site-directed mutagenesis. Available evidence indicates that the FAD chromophore of AppA may be protonated in the receptor state, and that it becomes exposed to solvent in the signaling state. Furthermore, experimental data lead to the suggestion that intramolecular proton transfer (that may involve [anionic] Tyr-17) forms the basis for the stabilization of the signaling state.     

Structures of the chromophore binding sites in BLUF domains as studied by molecular dynamics and quantum chemical calculations

BLUF (blue-light sensing using FAD) domains constitute a new family of flavin-based blue light photoreceptors. The photocycle of BLUF is unique in the sense that a few hydrogen bond rearrangements are accompanied by only slight structural changes in the bound chromophore. The hydrogen bond rearrangements upon illumination have been inferred from spectral changes in the chromophore: approximately 10 nm redshift of the absorption maximum and approximately 16 cm(-1) downshift of the C4=O stretching frequency. However, the exact features of the hydrogen bond network around the active site are still the subject of some controversy. In particular, the orientation of a conserved Gln (Gln63 in AppA) is presently one of the most questioned topics in the field. Here we perform molecular dynamics simulations for the wild-type AppA, AppA1-124C20S, BlrB and T110078 and furthermore quantum chemical calculations to investigate their spectroscopic properties in the dark and signaling states. On the basis of these results, we reveal the dynamic aspect of hydrogen bonding networks at the active site and propose theoretically reasonable models for the dark and signaling states of the BLUF domains.     

A Blue Light-inducible Phosphodiesterase Activity in the Cyanobacterium Synechococcus elongatus

A blue light-inducible phosphodiesterase (PDE) activity, specific for the hydrolysis of cyclic di-GMP (c-di-GMP), has been identified in a recombinant protein from Synechococcus elongatus. Blue light (BL) activation is accomplished by a light, oxygen, voltage (LOV) domain, found in plant phototropins and bacterial BL photoreceptors. The genome of S. elongatus contains two genes coding for proteins with LOV domains fused to EAL domains (SL1 and SL2). In both cases, a GGDEF motif is placed in between the LOV and the EAL motifs. Such arrangement is frequently found with diguanylate-cyclase (DGC) functions that form c-di-GMP. Cyclic di-GMP acts as a second messenger molecule regulating biofilm formation in many microbial species. Both enzyme activities modulate the intracellular level of this second messenger, although in most proteins only one of the two enzyme functions is active. Both S. elongatus LOV-GGDEF-EAL proteins were expressed in full length or as truncated proteins. Only the SL2 protein, expressed as a LOV-GGDEF-EAL construct, showed an increase of PDE activity upon BL irradiation, demonstrating this activity for the first time in a LOV-domain protein. Addition of GTP or c-di-GMP did not affect the observed enzymatic activity. In none of the full-length or truncated proteins was a DGC activity detected.     

Why BLUF photoreceptors with roseoflavin cofactors lose their biological functionality

The photophysics of roseoflavin in three different environments is investigated by using ab initio and quantum mechanics/molecular mechanics methods. Intramolecular charge transfer is shown to be responsible for the quenching of the fluorescence in the gas phase, and in the water environment. However, for the roseoflavin incorporated into the blue light using flavin (BLUF) protein environment (substituting the native flavin) no such deactivation is found. The conical intersection between the locally excited state of the chromophore and the charge transfer state involving the tyrosine residue, which in the native BLUF domain is responsible for initiating the photocycle, is missing for the roseoflavin substituted protein. This explains the experimental observations of the lack of any photocycle, and the loss of the biological function of the BLUF photoreceptor reported earlier.     

BLUF Hydrogen network dynamics and UV/Vis spectra: A combined molecular dynamics and quantum chemical study

Blue light sensing using flavin (BLUF) protein photoreceptor domains change their hydrogen bond network after photoexcitation. To explore this phenomenon, BLUF domains from R. sphaeroides were simulated using Amber99 molecular dynamics (MD). Five starting configurations were considered, to study different BLUF proteins (AppA/BlrB), Trp conformations (“W_in”/“W_out”), structure determination (X‐ray/NMR), and finally, His protonation states. We found dependencies of the hydrogen bonds on almost all parameters. Our data show an especially strong correlation of the Trp position and hydrogen bonds involving Gln63. The latter is in some contradiction to earlier results (Obanayama et al., Photochem. Photobiol. 2008, 84 10031010). Possible origins and implications are discussed. Our calculations support conjectures that Gln63 is more flexible with Trp104 in W_in position. Using snapshots from MD and time‐dependent density functional theory, UV/vis spectra for the chromophore were determined, which account for molecular motion of the protein under ambient conditions. In accord with experiment, it is found that the UV/vis spectra of BLUF bound flavin are red‐shifted and thermally broadened for all calculated π → π* transitions, relative to gas phase flavin at T = 0 K. However, differences in the spectra between the various BLUF configurations cannot be resolved with the present approach. © 2012 Wiley Periodicals, Inc.     

Escherichia coli: Dominance of Red Light over Other Visible Light Sources in Establishing Viable but Nonculturable State

In this study, the effect of UV-A and different wavelengths of visible light irradiations combined with or without a photosensitizer (methylene blue, MB) on the establishment of viable but nonculturable (VBNC) state in Escherichia coli was investigated. Survival of the E. coli was investigated by measuring plate counts, respiring cell count (RCC), direct viable count (DVC) and total counts over a period of up to 72 h. The inhibition rates of various light sources in the presence or absence of MB on E. coli in seawater were ranked in the order UV-A>red light>white light>blue light>green light (from greatest to least activation). E. coli survived for 10.2, 19.0, 21.3 and 24.04 h under exposure to red, white, blue and green light and for 6.8 h under exposure to UV-A in the presence of MB according to t ₉₉ . Although the VC declined to undetectable levels in a relatively short time, the RCC showed that some cells were still capable of respiration and, therefore, are assumed to have entered the VBNC phase. This is the first time that red light has been shown to have a stronger effect on E. coli survival and VBNC than white, green and blue light in seawater environment.     

Photodynamics of the small BLUF protein BlrB from Rhodobacter sphaeroides

The BLUF protein BlrB from the non-sulphur anoxyphototrophic purple bacterium Rhodobacter sphaeroides is characterized by absorption and emission spectroscopy. BlrB expressed from E. coli binding FAD, FMN, and riboflavin (called BrlB(I)) and recombinant BlrB containing only FAD (called BlrB(II)) are investigated. The dark-adapted proteins exist in two different receptor conformations (receptor states) with different sub-nanosecond fluorescence lifetimes (BLUF(r,f) and BLUF(r,sl)). Some of the flavin-cofactor (ca. 8%) is unbound in thermodynamic equilibrium with the bound cofactor. The two receptor conformations are transformed to putative signalling states (BLUF(s,f) and BLUF(s,sl)) of decreased fluorescence efficiency and shortened fluorescence lifetime by blue-light excitation. In the dark at room temperature both signalling states recover back to the initial receptor states with a time constant of about 2s. Quantum yields of signalling state formation of about 90% for BlrB(II) and about 40% for BlrB(I) were determined by intensity dependent transmission measurements. Extended blue-light excitation causes unbound flavin degradation (formation of lumichrome and lumiflavin-derivatives) and bound cofactor conversion to the semiquinone form. The flavin-semiquinone further reduces and the reduced flavin re-oxidizes back in the dark. A photo-dynamics scheme is presented and relevant quantum efficiencies and time constants are determined.     

Phototaxis in the cyanobacterium Synechocystis sp. PCC 6803: role of different photoreceptors

The second cyanobacterial phytochrome Cph2 from Synechocystis sp. PCC 6803 was suggested as a part of a light-stimulated signal transduction chain inhibiting movement toward blue light. Cph2 has the two bilin binding sites, cysteine-129 and cysteine-1022, that might be involved in sensing of red/far-red and blue light, respectively. Here, we present data on wavelength dependence of the phototaxis inhibition under blue light, indicating that Cph2 itself is the photoreceptor for this blue light response. We found that inhibition of blue-light phototaxis in wild-type cells occurred below the transition point of about 470 nm. Substitution of cysteine-1022 with valine led to photomovement of the cells toward blue light (cph2(-) mutant phenotype). Analysis of mutants lacking cysteine-129 in the N-terminal chromophore binding domain indicated that this domain is also important for Cph2 function or folding of the protein. Furthermore, putative blue-light and phytochrome-like photoreceptors encoded by the Synechocystis sp. PCC 6803 genome were inactivated in wild-type and cph2 knockout mutant background. Our results suggest that none of these potential photoreceptors interfere with Cph2 function, although inactivation of taxD1 as well as slr1694 encoding a BLUF protein led to cells that reversed the direction of movement under blue light illumination in mutant strains of cph2.