Spectroscopic and Computational Observation of Glutamine Tautomerization in the Blue Light Sensing Using Flavin Domain Photoreaction.

Blue light sensing using flavin (BLUF) domains constitute a family of flavin-binding photoreceptors of bacteria and eukaryotic algae. BLUF photoactivation proceeds via a light-driven hydrogen-bond switch among flavin adenine dinucleotide (FAD) and glutamine and tyrosine side chains, whereby FAD undergoes electron and proton transfer with tyrosine and is subsequently re-oxidized by a hydrogen back-shuttle in picoseconds, constituting an important model system to understand proton-coupled electron transfer in biology. The specific structure of the hydrogen-bond patterns and the prevalence of glutamine tautomeric states in dark-adapted (DA) and light-activated (LA) states have remained controversial. Here, we present a combined femtosecond stimulated Raman spectroscopy (FSRS), computational chemistry, and site-selective isotope labeling Fourier-transform infrared spectroscopy (FTIR) study of the Slr1694 BLUF domain. FSRS showed distinct vibrational bands from the FADS1 singlet excited state. We observed small but significant shifts in the excited-state vibrational frequency patterns of the DA and LA states, indicating that these frequencies constitute a sensitive probe for the hydrogen-bond arrangement around FAD. Excited-state model calculations utilizing four different realizations of hydrogen bond patterns and glutamine tautomeric states were consistent with a BLUF reaction model that involved glutamine tautomerization to imidic acid, accompanied by a rotation of its side chain. A combined FTIR and double-isotope labeling study, with 13C labeling of FAD and 15N labeling of glutamine, identified the glutamine imidic acid C═N stretch vibration in the LA state and the Gln C═O in the DA state. Hence, our study provides support for glutamine tautomerization and side-chain rotation in the BLUF photoreaction.

Helical Contributions Mediate Light-Activated Conformational Change in the LOV2 Domain of Avena sativa Phototropin 1.

Algae, plants, bacteria, and fungi contain flavin-binding light-oxygen-voltage (LOV) domains that function as blue light sensors to control cellular responses to light. In the second LOV domain of phototropins, called LOV2 domains, blue light illumination leads to covalent bond formation between protein and flavin that induces the dissociation and unfolding of a C-terminally attached α helix (Jα) and the N-terminal helix (A'α). To date, the majority of studies on these domains have focused on versions that contain truncations in the termini, which creates difficulties when extrapolating to the much larger proteins that contain these domains. Here, we study the influence of deletions and extensions of the A'α helix of the LOV2 domain of Avena sativa phototropin 1 (AsLOV2) on the light-triggered structural response of the protein by Fourier-transform infrared difference spectroscopy. Deletion of the A'α helix abolishes the light-induced unfolding of Jα, whereas extensions of the A'α helix lead to an attenuated structural change of Jα. These results are different from shorter constructs, indicating that the conformational changes in full-length phototropin LOV domains might not be as large as previously assumed, and that the well-characterized full unfolding of the Jα helix in AsLOV2 with short A'α helices may be considered a truncation artifact. It also suggests that the N- and C-terminal helices of phot-LOV2 domains are necessary for allosteric regulation of the phototropin kinase domain and may provide a basis for signal integration of LOV1 and LOV2 domains in phototropins.

Reaction dynamics of the chimeric channelrhodopsin C1C2.

Channelrhodopsin (ChR) is a key protein of the optogenetic toolkit. C1C2, a functional chimeric protein of Chlamydomonas reinhardtii ChR1 and ChR2, is the only ChR whose crystal structure has been solved, and thus uniquely suitable for structure-based analysis. We report C1C2 photoreaction dynamics with ultrafast transient absorption and multi-pulse spectroscopy combined with target analysis and structure-based hybrid quantum mechanics/molecular mechanics calculations. Two relaxation pathways exist on the excited (S1) state through two conical intersections CI1 and CI2, that are reached via clockwise and counter-clockwise rotations: (i) the C13=C14 isomerization path with 450 fs via CI1 and (ii) a relaxation path to the initial ground state with 2.0 ps and 11 ps via CI2, depending on the hydrogen-bonding network, hence indicating active-site structural heterogeneity. The presence of the additional conical intersection CI2 rationalizes the relatively low quantum yield of photoisomerization (30 ± 3%), reported here. Furthermore, we show the photoreaction dynamics from picoseconds to seconds, characterizing the complete photocycle of C1C2.

Light-Induced Conformational Changes in the Plant Cryptochrome Photolyase Homology Region Resolved by Selective Isotope Labeling and Infrared Spectroscopy.

Plant cryptochromes are photoreceptors that regulate flowering, circadian rhythm and photomorphogenesis in response to blue and UV-A light. It has been demonstrated that the oxidized flavin cofactor is photoreduced to the neutral radical state via separate electron and proton transfer. Conformational changes have been found in the C-terminal extension, but few studies have addressed the changes in secondary structure in the sensory photolyase homology region (PHR). Here, we investigated the PHR of the plant cryptochrome from the green alga Chlamydomonas reinhardtii by light-induced infrared difference spectroscopy in combination with global 13 C and 15 N isotope labeling. Assignment of the signals is achieved by establishing a labeling strategy for cryptochromes that preserves the flavin at natural abundance. We demonstrate by UV/vis spectroscopy that the integrity of the sample is maintained and by mass spectrometry that the global labeling was highly efficient. As a result, difference bands are resolved at full intensity that at natural abundance are compensated by the overlap of flavin and protein signals. These bands are assigned to prominent conformational changes in the PHR by blue light illumination. We postulate that not only the partial unfolding of the C-terminal extension but also changes in the PHR may mediate signaling events.

Photoadduct Formation from the FMN Singlet Excited State in the LOV2 Domain of Chlamydomonas reinhardtii Phototropin.

The two light, oxygen, and voltage domains of phototropin are blue-light photoreceptor domains that control various functions in plants and green algae. The key step of the light-driven reaction is the formation of a photoadduct between its FMN chromophore and a conserved cysteine, where the canonical reaction proceeds through the FMN triplet state. Here, complete photoreaction mapping of CrLOV2 from Chlamydomonas reinhardtii phototropin and AsLOV2 from Avena sativa phototropin-1 was realized by ultrafast broadband spectroscopy from femtoseconds to microseconds. We demonstrate that in CrLOV2, a direct photoadduct formation channel originates from the initially excited singlet state, in addition to the canonical reaction through the triplet state. This direct photoadduct reaction is coupled by a proton or hydrogen transfer process, as indicated by a significant kinetic isotope effect of 1.4 on the fluorescence lifetime. Kinetic model analyses showed that 38% of the photoadducts are generated from the singlet excited state.

Unfolding of the C-Terminal Jα Helix in the LOV2 Photoreceptor Domain Observed by Time-Resolved Vibrational Spectroscopy.

Light-triggered reactions of biological photoreceptors have gained immense attention for their role as molecular switches in their native organisms and for optogenetic application. The light, oxygen, and voltage 2 (LOV2) sensing domain of plant phototropin binds a C-terminal Jα helix that is docked on a ß-sheet and unfolds upon light absorption by the flavin mononucleotide (FMN) chromophore. In this work, the signal transduction pathway of LOV2 from Avena sativa was investigated using time-resolved infrared spectroscopy from picoseconds to microseconds. In D2O buffer, FMN singlet-to-triplet conversion occurs in 2 ns and formation of the covalent cysteinyl-FMN adduct in 10 µs. We observe a two-step unfolding of the Jα helix: The first phase occurs concomitantly with Cys-FMN covalent adduct formation in 10 µs, along with hydrogen-bond rupture of the FMN C4═O with Gln-513, motion of the ß-sheet, and an additional helical element. The second phase occurs in approximately 240 µs. The final spectrum at 500 µs is essentially identical to the steady-state light-minus-dark Fourier transform infrared spectrum, indicating that Jα helix unfolding is complete on that time scale.

Natural Resources for Optogenetic Tools.

Photoreceptors are found in all kingdoms of life and mediate crucial responses to environmental challenges. Nature has evolved various types of photoresponsive protein structures with different chromophores and signaling concepts for their given purpose. The abundance of these signaling proteins as found nowadays by (meta-)genomic screens enriched the palette of optogenetic tools significantly. In addition, molecular insights into signal transduction mechanisms and design principles from biophysical studies and from structural and mechanistic comparison of homologous proteins opened seemingly unlimited possibilities for customizing the naturally occurring proteins for a given optogenetic task. Here, a brief overview on the photoreceptor concepts already established as optogenetic tools in natural or engineered form, their photochemistry and their signaling/design principles is given. Finally, so far not regarded photosensitive modules and protein architectures with potential for optogenetic application are described.

Light-Induced Rearrangement of the ß5 Strand in the BLUF Photoreceptor SyPixD (Slr1694).

The structural changes that facilitate signal transduction in blue light sensors using FAD (BLUF) photoreceptors and confer the stability of the rearranged hydrogen bond network between flavin and protein in the signaling state are still poorly understood. Here, we investigate a semiconserved Trp residue in SyPixD (Slr1694) by isotope-edited vibrational spectroscopy and site-directed mutagenesis. In the signaling state, a ß-sheet structure involving the backbone of W91 is formed without apparent change of environment of the W91 indole side chain. Mutation of W91, however, significantly influences the stability of the light-adapted state, suggesting that backbone rigidity rather than discrete side-chain conformations govern the stability of the light-adapted state. On the basis of computational and crystallographic models, we interpret these changes as a +1 register shift of the ß2/ß5 interaction with an unaffected indole side-chain conformation, rather than a +2 register shift accompanied by an indole side-chain flip that was previously proposed on the basis of X-ray structures.

A proposal for a dipole-generated BLUF domain mechanism.

The resting and signaling structures of the blue-light sensing using flavin (BLUF) photoreceptor domains are still controversially debated due to differences in the molecular models obtained by crystal and NMR structures. Photocycles for the given preferred structural framework have been established, but a unifying picture combining experiment and theory remains elusive. We summarize present work on the AppA BLUF domain from both experiment and theory. We focus on IR and UV/vis spectra, and to what extent theory was able to reproduce experimental data and predict the structural changes upon formation of the signaling state. We find that the experimental observables can be theoretically reproduced employing any structural model, as long as the orientation of the signaling essential Gln63 and its tautomer state are a choice of the modeler. We also observe that few approaches are comparative, e.g., by considering all structures in the same context. Based on recent experimental findings and a few basic calculations, we suggest the possibility for a BLUF activation mechanism that only relies on electron transfer and its effect on the local electrostatics, not requiring an associated proton transfer. In this regard, we investigate the impact of dispersion correction on the interaction energies arising from weakly bound amino acids.

Femto- to Microsecond Photodynamics of an Unusual Bacteriophytochrome.

A bacteriophytochrome from Stigmatella aurantiaca is an unusual member of the bacteriophytochrome family that is devoid of hydrogen bonding to the carbonyl group of ring D of the biliverdin (BV) chromophore. The photodynamics of BV in SaBphP1 wild type and the single mutant T289H reintroducing hydrogen bonding to ring D show that the strength of this particular weak interaction determines excited-state lifetime, Lumi-R quantum yield, and spectral heterogeneity. In particular, excited-state decay is faster in the absence of hydrogen-bonding to ring D, with excited-state half-lives of 30 and 80 ps for wild type and the T289H mutant, respectively. Concomitantly, the Lumi-R quantum yield is two times higher in wild type as compared with the T289H mutant. Furthermore, the spectral heterogeneity in the wild type is significantly higher than that in the T289H mutant. By extending the observable time domain to 25 µs, we observe a new deactivation pathway from the Lumi-R intermediate in the 100 ns time domain that corresponds to a backflip of ring D to the original Pr 15Za isomeric state.

Proton-Coupled Electron Transfer Constitutes the Photoactivation Mechanism of the Plant Photoreceptor UVR8.

UVR8 is a novel UV-B photoreceptor that regulates a range of plant responses and is already used as a versatile optogenetic tool. Instead of an exogenous chromophore, UVR8 uniquely employs tryptophan side chains to accomplish UV-B photoreception. UV-B absorption by homodimeric UVR8 induces monomerization and hence signaling, but the underlying photodynamic mechanisms are not known. Here, by using a combination of time-resolved fluorescence and absorption spectroscopy from femto- to microseconds, we provide the first experimental evidence for the UVR8 molecular signaling mechanism. The results indicate that tryptophan residues at the dimer interface engage in photoinduced proton coupled electron transfer reactions that induce monomerization.

Photoinduced formation of flavin radicals in BLUF domains lacking the central glutamine.

Blue light receptors using FAD (BLUFs) facilitate blue light-induced signal transduction via light-induced rearrangement of hydrogen bonds between the flavin chromophore and a conserved glutamine side chain. Here, we investigated the photochemistry of the BLUF domain Slr1694 from Synechocystis sp. in which the glutamine side chain was removed. Without the glutamine, no red-shifted signaling state is formed, but light-induced proton-coupled electron transfer between protein and flavin takes place similarly as for the wild-type protein. However, the lifetime of the neutral flavin semiquinone-tyrosyl radical pair is greatly prolonged from < 100 ps to several nanoseconds, which indicates that the formation of radical intermediates drives the hydrogen bond rearrangement in BLUF photoactivation. Moreover, glutamine plays a central role in the molecular organization of the hydrogen bond network in the flavin-binding pocket, as its removal enhances electron transfer from tyrosine to the excited flavin, and enables competing electron transfer from a nearby tryptophan.

Photoinduced transformation of UVR8 monitored by vibrational and fluorescence spectroscopy.

Tryptophan residues at the dimer interface of the plant photoreceptor UVR8 promote monomerisation after UV-B absorption via a so far unknown mechanism. Using FTIR spectroscopy we assign light-induced structural transitions of UVR8 mainly to amino acid side chains without major transformations of the secondary structure of the physiologically relevant C-terminal extension. Additionally, we assign the monomerisation associated increase and red shift of the UVR8 tryptophan emission to a photoinduced rearrangement of tryptophan side chains and a relocation of the aspartic acid residues D96 and D107, respectively. By illumination dependent emission spectroscopy we furthermore determined the quantum yield of photoinduced monomerisation to 20 ± 8%.

Upgrading a microplate reader for photobiology and all-optical experiments.

Automation can vastly reduce the cost of experimental labor and thus facilitate high experimental throughput, but little off-the-shelf hardware for the automation of illumination experiments is commercially available. Here, we use inexpensive open-source electronics to add programmable illumination capabilities to a multimode microplate reader. We deploy this setup to characterize light-triggered phenomena in three different sensory photoreceptors. First, we study the photoactivation of Arabidopsis thaliana phytochrome B by light of different wavelengths. Second, we investigate the dark-state recovery kinetics of the Synechocystis sp. blue-light sensor Slr1694 at multiple temperatures and imidazole concentrations; while the kinetics of the W91F mutant of Slr1694 are strongly accelerated by imidazole, the wild-type protein is hardly affected. Third, we determine the light response of the Beggiatoa sp. photoactivatable adenylate cyclase bPAC in Chinese hamster ovary cells. bPAC is activated by blue light in dose-dependent manner with a half-maximal intensity of 0.58 mW cm(-2); intracellular cAMP spikes generated upon bPAC activation decay with a half time of about 5 minutes after light switch-off. Taken together, we present a setup which is easily assembled and which thus offers a facile approach to conducting illumination experiments at high throughput, reproducibility and fidelity.

Flavin adenine dinucleotide chromophore charge controls the conformation of cyclobutane pyrimidine dimer photolyase α-helices.

Observations of light-receptive enzyme complexes are usually complicated by simultaneous overlapping signals from the chromophore, apoprotein, and substrate, so that only the initial, ultrafast, photon-chromophore reaction and the final, slow, protein conformational change provide separate, nonoverlapping signals. Each provides its own advantages, whereas sometimes the overlapping signals from the intervening time scales still cannot be fully deconvoluted. We overcome the problem by using a novel method to selectively isotope-label the apoprotein but not the flavin adenine dinucleotide (FAD) cofactor. This allowed the Fourier transform infrared (FTIR) signals to be separated from the apoprotein, FAD cofactor, and DNA substrate. Consequently, a comprehensive structure-function study by FTIR spectroscopy of the Escherichia coli cyclobutane pyrimidine dimer photolyase (CPD-PHR) DNA repair enzyme was possible. FTIR signals could be identified and assigned upon FAD photoactivation and DNA repair, which revealed protein dynamics for both processes beyond simple one-electron reduction and ejection, respectively. The FTIR data suggest that the synergistic cofactor-protein partnership in CPD-PHR linked to changes in the shape of FAD upon one-electron reduction may be coordinated with conformational changes in the apoprotein, allowing it to fit the DNA substrate. Activation of the CPD-PHR chromophore primes the apoprotein for subsequent DNA repair, suggesting that CPD-PHR is not simply an electron-ejecting structure. When FAD is activated, changes in its structure may trigger coordinated conformational changes in the apoprotein and thymine carbonyl of the substrate, highlighting the role of Glu275. In contrast, during DNA repair and release processes, primary conformational changes occur in the enzyme and DNA substrate, with little contribution from the FAD cofactor and surrounding amino acid residues.

Absorption and emission spectroscopic characterization of photo-dynamics of photoactivated adenylyl cyclase mutant bPAC-Y7F of Beggiatoa sp.

The photoactivated cyclase bPAC of the microbial mats bacterium Beggiatoa sp. consists of a BLUF domain and an adenylyl cyclase domain. It has strong activity of photo-induced cyclic adenylyl monophosphate (cAMP) formation and is therefore an important optogenetic tool in neuroscience applications. The SUMO-bPAC-Y7F mutant where Tyr-7 is replaced by Phe-7 in the BLUF domain has lost the typical BLUF domain photo-cycle dynamics. Instead, the investigated SUMO-bPAC-Y7F mutant consisted of three protein conformations with different triplet based photo-dynamics: (i) reversible flavin quinone (Fl) cofactor reduction to flavin semiquinone (FlH), (ii) reversible violet/near ultraviolet absorbing flavin photoproduct (FlA) formation, and (iii) irreversible red absorbing flavin photoproduct (FlC) formation. Absorption and emission spectroscopic measurements on SUMO-bPAC-Y7F were carried out before, during and after light exposure. Flavin photo-dynamics schemes are developed for the SUMO-bPAC-Y7F fractions performing photo-induced FlH, FlA, and FlC formation. Quantitative parameters of the flavin cofactor excitation, relaxation and recovery dynamics in SUMO-bPAC-Y7F are determined.

Key residues for the light regulation of the blue light-activated adenylyl cyclase from Beggiatoa sp.

Photoactivated adenylyl cyclases are powerful tools for optogenetics and for investigating signal transduction mechanisms in biological photoreceptors. Because of its large increase in enzyme activity in the light, the BLUF (blue light sensor using flavin adenine dinucleotide)-activated adenylyl cyclase (bPAC) from Beggiatoa sp. is a highly attractive model system for studying BLUF domain signaling. In this report, we studied the influence of site-directed mutations within the BLUF domain on the light regulation of the cyclase domain and determined key elements for signal transduction and color tuning. Photoactivation of the cyclase domain is accomplished via strand ß5 of the BLUF domain and involves the formation of helical structures in the cyclase domain as assigned by vibrational spectroscopy. In agreement with earlier studies, we observed severely impaired signaling in mutations directly on strand ß5 as well as in mutations affecting the hydrogen bond network around the flavin. Moreover, we identified a bPAC mutant with red-shifted absorbance and a decreased dark activity that is highly valuable for long-term optogenetic experiments. Additionally, we discovered a mutant that forms a stable neutral flavin semiquinone radical in the BLUF domain and surprisingly exhibits an inversion of light activation.

Photoactivation mechanisms of flavin-binding photoreceptors revealed through ultrafast spectroscopy and global analysis methods.

Flavin-binding photoreceptor proteins use the isoalloxazine moiety of flavin cofactors to absorb light in the blue/UV-A wavelength region and subsequently translate it into biological information. The underlying photochemical reactions and protein structural dynamics are delicately tuned by the protein environment and represent fundamental reactions in biology and chemistry. Due to their photo-switchable nature, these proteins can be studied efficiently with laser-flash induced transient absorption and emission spectroscopy with temporal precision down to the femtosecond time domain. Here, we describe the application of both visible and mid-IR ultrafast transient absorption and time-resolved fluorescence methods in combination with sophisticated global analysis procedures to elucidate the photochemistry and signal transduction of BLUF (Blue light receptors using FAD) and LOV (Light oxygen voltage) photoreceptor domains.

Bistable retinal schiff base photodynamics of histidine kinase rhodopsin HKR1 from Chlamydomonas reinhardtii.

The photodynamics of the recombinant rhodopsin fragment of the histidine kinase rhodopsin HKR1 from Chlamydomonas reinhardtii was studied by absorption and fluorescence spectroscopy. The retinal cofactor of HKR1 exists in two Schiff base forms RetA and RetB. RetA is the deprotonated 13-cis-retinal Schiff base (RSB) absorbing in the UVA spectral region. RetB is the protonated all-trans RSB absorbing in the blue spectral region. Blue light exposure converts RetB fully to RetA. UVA light exposure converts RetA to RetB and RetB to RetA giving a mixture determined by their absorption cross sections and their conversion efficiencies. The quantum efficiencies of conversion of RetA to RetB and RetB to RetA were determined to be 0.096 ± 0.005 and 0.405 ± 0.01 respectively. In the dark thermal equilibration between RetA and RetB with dominant RetA content occurred with a time constant of about 3 days at room temperature. The fluorescence emission behavior of RetA and RetB was studied, and fluorescence quantum yields of ϕ(F) (RetA) = 0.00117 and ϕ(F) (RetB) = 9.4 × 10(-5) were determined. Reaction coordinate schemes of the photodynamics are developed.