Elucidating the Signal Transduction Mechanism of the Blue-Light-Regulated Photoreceptor YtvA: From Photoactivation to Downstream Regulation.

The blue-light photoreceptor YtvA from Bacillus subtilis has an N-terminal flavin mononucleotide (FMN)-binding light-oxygen-voltage (LOV) domain that is fused to a C-terminal sulfate transporter and anti-σ factor antagonist (STAS) output domain. To interrogate the signal transduction pathway that leads to photoactivation, the STAS domain was replaced with a histidine kinase, so that photoexcitation of the flavin could be directly correlated with biological activity. N94, a conserved Asn that is hydrogen bonded to the FMN C2═O group, was replaced with Ala, Asp, and Ser residues to explore the role of this residue in triggering the structural dynamics that activate the output domain. Femtosecond to millisecond time-resolved multiple probe spectroscopy coupled with a fluorescence polarization assay revealed that the loss of the hydrogen bond between N94 and the C2═O group decoupled changes in the protein structure from photoexcitation. In addition, alterations in N94 also decreased the stability of the Cys-FMN adduct formed in the light-activated state by up to a factor of ∼25. Collectively, these studies shed light on the role of the hydrogen bonding network in the LOV ß-scaffold in signal transduction.

When microplastics are not plastic: Chemical characterization of environmental microfibers using stimulated Raman microspectroscopy.

The abundance of anthropogenic debris dispersed in the environment is exponentially growing, raising concerns about marine life and human exposure to microplastics. Microfibers are the most abundant microplastic type in the environment. However, recent research suggests that most microfibers dispersed in the environment are not made of synthetic polymers. In this work, we systematically tested this assumption by determining the man-made or natural origin of microfibers found in different environments, including surface waters, sediments at depths >5000 m and highly sensitive habitats like mangroves and seagrass, and treated water using stimulated Raman scattering (SRS) microscopy. Our findings show that ¾th of analyzed microfibers are of natural origin. One plastic fiber is estimated per every 50 L in surface seawater, every 5 L in desalinated drinking water, every 3 g in deep sea sediments and every 27 g in coastal sediments. Synthetic fibers were significantly larger in surface seawaters compared to organic fibers due to higher resistance to solar radiation. These results emphasize the necessity of using spectroscopical methods to assess the origin of environmental microfibers to accurately estimate the abundance of synthetic materials in the environment.

Frequency-modulation stimulated Raman scattering microscopy with an acousto-optic tunable filter.

Stimulated Raman scattering (SRS) microscopy is increasingly employed for highly specific, label-free, and high-speed bioimaging. Despite its benefits, SRS is susceptible to spurious background signals caused by competing effects, which lower the possible imaging contrast and sensitivity. An efficient approach to suppress these undesired background signals is frequency-modulation (FM) SRS, which exploits the competing effects' weak spectral dependence compared to the SRS signal's high spectral specificity. We propose an FM-SRS scheme realized with an acousto-optic tunable filter, which presents a few advantages compared to other solutions presented in the literature. In particular, it can perform automated measurements from the fingerprint to the CH-stretching region of the vibrational spectrum without any manual adjustment of the optical setup. Moreover, it allows simple all-electronic control of the spectral separation and relative intensities of the pair of probed wavenumbers.

Spectrochemistry of Firefly Bioluminescence.

The chemical reactions underlying the emission of light in fireflies and other bioluminescent beetles are some of the most thoroughly studied processes by scientists worldwide. Despite these remarkable efforts, fierce academic arguments continue around even some of the most fundamental aspects of the reaction mechanism behind the beetle bioluminescence. In an attempt to reach a consensus, we made an exhaustive search of the available literature and compiled the key discoveries on the fluorescence and chemiluminescence spectrochemistry of the emitting molecule, the firefly oxyluciferin, and its chemical analogues reported over the past 50+ years. The factors that affect the light emission, including intermolecular interactions, solvent polarity, and electronic effects, were analyzed in the context of both the reaction mechanism and the different colors of light emitted by different luciferases. The collective data points toward a combined emission of multiple coexistent forms of oxyluciferin as the most probable explanation for the variation in color of the emitted light. We also highlight realistic research directions to eventually address some of the remaining questions related to firefly bioluminescence. It is our hope that this extensive compilation of data and detailed analysis will not only consolidate the existing body of knowledge on this important phenomenon but will also aid in reaching a wider consensus on some of the mechanistic details of firefly bioluminescence.

Hadamard-transform spectral acquisition with an acousto-optic tunable filter in a broadband stimulated Raman scattering microscope.

We present a novel configuration for high spectral resolution multiplexing acquisition based on the Hadamard transform in stimulated Raman scattering (SRS) microscopy. The broadband tunable output of a dual-beam femtosecond laser is filtered by a fast, narrowband, and multi-channel acousto-optic tunable filter (AOTF). By turning on and off different subsets of its 8 independent channels, the AOTF generates the spectral masks given by the Hadamard matrix. We demonstrate a seamless and automated operation in the Raman fingerprint and CH-stretch regions. In the presence of additive noise, the spectral measurements using the multiplexed method show the same signal-to-noise ratio of conventional single-wavenumber acquisitions performed with 4 times longer integration time.

Stimulated Raman microspectroscopy as a new method to classify microfibers from environmental samples.

Microfibers are reported as the most abundant microparticle type in the environment. Their small size and light weight allow easy and fast distribution, but also make it challenging to determine their chemical composition. Vibrational microspectroscopy methods as infrared and spontaneous Raman microscopy have been widely used for the identification of environmental microparticles. However, only few studies report on the identification of microfibers, mainly due to difficulties caused by their small diameter. Here we present the use of Stimulated Raman Scattering (SRS) microscopy for fast and reliable classification of microfibers from environmental samples. SRS microscopy features high sensitivity and has the potential to be faster than other vibrational microspectroscopy methods. As a proof of principle, we analyzed fibers extracted from the fish gastrointestinal (GIT) tract, deep-sea and coastal sediments, surface seawater and drinking water. Challenges were faced while measuring fibers from the fish GIT, due to the acidic degradation they undergo. However, the main vibrational peaks were still recognizable and sufficient to determine the natural or synthetic origin of the fibers. Notably, our results are in accordance to other recent studies showing that the majority of the analyzed environmental fibers has a natural origin. Our findings suggest that advanced spectroscopic methods must be used for estimation of the plastic fibers concentration in the environment.

Time-resolved photoluminescence of 6-thienyl-lumazine fluorophores in cellulose acetate nanofibers for detection of mercury ions.

Time-resolved photoluminescence measurements were used to characterize the photophysical properties of 6-thienyllumazine (TLm) fluorophores in cellulose acetate nanofibers (NFs) in the presence and absence of mercuric acetate salts. In solution, excited-state proton transfer (ESPT) from TLm to water molecules was investigated at pH from 2 to 12. The insertion of thienyl group into lumazine introduces cis and trans conformers while keeping the same tautomerization structures. Global and target analyses were employed to resolve the true emission spectra of all prototropic, tautomeric, and rotameric species for TLm in water. The results support the premise that only the cis conformers are related to the ESPT process. However, no ESPT from TLm to a nearby water molecule was observed in NFs. The addition of NFs increases the excited-state lifetime of TLm in the solid state because of combined polarity/confinement effects. The solid-state fluorescence of TLm (in NFs) was quenched by mercuric acetate through different mechanisms-dynamic and static-depending on the applied pressure-atmospheric and vacuum, respectively. The new solid-state sensor is simple, ecofriendly, and instantly fabricated. TLM-loaded NFs can detect mercuric ions at a concentration of 50 picomolar. The formation of non-fluorescent ground-state complex between TLm molecules and mercuric ions inside the pores of NFs was achieved under vacuum condition.

Fingerprint-to-CH stretch continuously tunable high spectral resolution stimulated Raman scattering microscope.

Stimulated Raman scattering (SRS) microscopy is a label-free method generating images based on chemical contrast within samples, and has already shown its great potential for high-sensitivity and fast imaging of biological specimens. The capability of SRS to collect molecular vibrational signatures in bio-samples, coupled with the availability of powerful statistical analysis methods, allows quantitative chemical imaging of live cells with sub-cellular resolution. This application has substantially driven the development of new SRS microscopy platforms. Indeed, in recent years, there has been a constant effort on devising configurations able to rapidly collect Raman spectra from samples over a wide vibrational spectral range, as needed for quantitative analysis by using chemometric methods. In this paper, an SRS microscope which exploits spectral shaping by a narrowband and rapidly tunable acousto-optical tunable filter (AOTF) is presented. This microscope enables spectral scanning from the Raman fingerprint region to the Carbon-Hydrogen (CH)-stretch region without any modification of the optical setup. Moreover, it features also a high enough spectral resolution to allow resolving Raman peaks in the crowded fingerprint region. Finally, application of the developed SRS microscope to broadband hyperspectral imaging of biological samples over a large spectral range from 800 to 3600 cm-1 , is demonstrated.

Infrared spectroscopy reveals multi-step multi-timescale photoactivation in the photoconvertible protein archetype dronpa.

Photochromic fluorescent proteins play key roles in super-resolution microscopy and optogenetics. The light-driven structural changes that modulate the fluorescence involve both trans-to-cis isomerization and proton transfer. The mechanism, timescale and relative contribution of chromophore and protein dynamics are currently not well understood. Here, the mechanism of off-to-on-state switching in dronpa is studied using femtosecond-to-millisecond time-resolved infrared spectroscopy and isotope labelling. Chromophore and protein dynamics are shown to occur on multiple timescales, from picoseconds to hundreds of microseconds. Following excitation of the trans chromophore, a ground-state primary product is formed within picoseconds. Surprisingly, the characteristic vibrational spectrum of the neutral cis isomer appears only after several tens of nanoseconds. Further fluctuations in protein structure around the neutral cis chromophore are required to form a new intermediate, which promotes the final proton-transfer reaction. These data illustrate the interplay between chromophore dynamics and the protein environment underlying fluorescent protein photochromism.

Turning on Solid-State Fluorescence with Light.

A bioinspired fluorophore that is analogous to the substrate in the bioluminescence of fireflies was prepared and reacts when exposed to weak blue LED light. Upon excitation, this material is photodecarboxylated with a nearly 81-fold enhancement of the solid-state emission, the fluorescence quantum yield of the product in solution is approximately 90 %, and violent disintegrative effects occur as a result of the release of carbon dioxide. Crystallographic and computational results, together with global spectral analysis of the kinetics, confirmed that most of the emission observed in the decay-associated spectra is intrinsic to the product molecule, with only a minor contribution from an excimer through π-π stacking of the molecules in the crystal.

Variation in LOV Photoreceptor Activation Dynamics Probed by Time-Resolved Infrared Spectroscopy.

The light, oxygen, voltage (LOV) domain proteins are blue light photoreceptors that utilize a noncovalently bound flavin mononucleotide (FMN) cofactor as the chromophore. The modular nature of these proteins has led to their wide adoption in the emerging fields of optogenetics and optobiology, where the LOV domain has been fused to a variety of output domains leading to novel light-controlled applications. In this work, we extend our studies of the subpicosecond to several hundred microsecond transient infrared spectroscopy of the isolated LOV domain AsLOV2 to three full-length photoreceptors in which the LOV domain is fused to an output domain: the LOV-STAS protein, YtvA, the LOV-HTH transcription factor, EL222, and the LOV-histidine kinase, LovK. Despite differences in tertiary structure, the overall pathway leading to cysteine adduct formation from the FMN triplet state is highly conserved, although there are slight variations in rate. However, significant differences are observed in the vibrational spectra and kinetics after adduct formation, which are directly linked to the specific output function of the LOV domain. While the rate of adduct formation varies by only 3.6-fold among the proteins, the subsequent large-scale structural changes in the full-length LOV photoreceptors occur over the micro- to submillisecond time scales and vary by orders of magnitude depending on the different output function of each LOV domain.

Photoactivation of the BLUF Protein PixD Probed by the Site-Specific Incorporation of Fluorotyrosine Residues.

The flavin chromophore in blue-light-using FAD (BLUF) photoreceptors is surrounded by a hydrogen bond network that senses and responds to changes in the electronic structure of the flavin on the ultrafast time scale. The hydrogen bond network includes a strictly conserved Tyr residue, and previously we explored the role of this residue, Y21, in the photoactivation mechanism of the BLUF protein AppABLUF by the introduction of fluorotyrosine (F-Tyr) analogues that modulated the pKa and reduction potential of Y21 by 3.5 pH units and 200 mV, respectively. Although little impact on the forward (dark- to light-adapted form) photoreaction was observed, the change in Y21 pKa led to a 4000-fold increase in the rate of dark-state recovery. In the present work we have extended these studies to the BLUF protein PixD, where, in contrast to AppABLUF, modulation in the Tyr (Y8) pKa has a profound impact on the forward photoreaction. In particular, a decrease in Y8 pKa by 2 or more pH units prevents formation of a stable light state, consistent with a photoactivation mechanism that involves proton transfer or proton-coupled electron transfer from Y8 to the electronically excited FAD. Conversely, the effect of pKa on the rate of dark recovery is markedly reduced in PixD. These observations highlight very significant differences between the photocycles of PixD and AppABLUF, despite their sharing highly conserved FAD binding architectures.

Ultrafast Excited State Dynamics in Molecular Motors: Coupling of Motor Length to Medium Viscosity.

Photochemically driven molecular motors convert the energy of incident radiation to intramolecular rotational motion. The motor molecules considered here execute four step unidirectional rotational motion. This comprises a pair of successive light induced isomerizations to a metastable state followed by thermal helix inversions. The internal rotation of a large molecular unit required in these steps is expected to be sensitive to both the viscosity of the medium and the volume of the rotating unit. In this work, we describe a study of motor motion in both ground and excited states as a function of the size of the rotating units. The excited state decay is ultrafast, highly non-single exponential, and is best described by a sum of three exponential relaxation components. The average excited state decay time observed for a series of motors with substituents of increasing volume was determined. While substitution does affect the lifetime, the size of the substituent has only a minor effect. The solvent polarity dependence is also slight, but there is a significant solvent viscosity effect. Increasing the viscosity has no effect on the fastest of the three decay components, but it does lengthen the two slower decay times, consistent with them being associated with motion along an intramolecular coordinate displacing a large solvent volume. However, these slower relaxation times are again not a function of the size of the substituent. We conclude that excited state decay arises from motion along a coordinate which does not necessarily require complete rotation of the substituents through the solvent, but is instead more localized in the core structure of the motor. The decay of the metastable state to the ground state through a helix inversion occurs 14 orders of magnitude more slowly than the excited state decay, and was measured as a function of substituent size, solvent viscosity and temperature. In this case neither substituent size nor solvent viscosity influences the rate, which is entirely determined by the activation barrier. This result is different to similar studies of an earlier generation of molecular motors, which suggests different microscopic mechanisms are in operation in the different generations. Finally, the rate of photochemical isomerization was studied for the series of motors, and those with the largest volume substituents showed the highest photochemical cross section.

Femtosecond to Millisecond Dynamics of Light Induced Allostery in the Avena sativa LOV Domain.

The rational engineering of photosensor proteins underpins the field of optogenetics, in which light is used for spatiotemporal control of cell signaling. Optogenetic elements function by converting electronic excitation of an embedded chromophore into structural changes on the microseconds to seconds time scale, which then modulate the activity of output domains responsible for biological signaling. Using time-resolved vibrational spectroscopy coupled with isotope labeling, we have mapped the structural evolution of the LOV2 domain of the flavin binding phototropin Avena sativa (AsLOV2) over 10 decades of time, reporting structural dynamics between 100 fs and 1 ms after optical excitation. The transient vibrational spectra contain contributions from both the flavin chromophore and the surrounding protein matrix. These contributions are resolved and assigned through the study of four different isotopically labeled samples. High signal-to-noise data permit the detailed analysis of kinetics associated with the light activated structural evolution. A pathway for the photocycle consistent with the data is proposed. The earliest events occur in the flavin binding pocket, where a subpicosecond perturbation of the protein matrix occurs. In this perturbed environment, the previously characterized reaction between triplet state isoalloxazine and an adjacent cysteine leads to formation of the adduct state; this step is shown to exhibit dispersive kinetics. This reaction promotes coupling of the optical excitation to successive time-dependent structural changes, initially in the ß-sheet and then α-helix regions of the AsLOV2 domain, which ultimately gives rise to Jα-helix unfolding, yielding the signaling state. This model is tested through point mutagenesis, elucidating in particular the key mediating role played by Q513.

Excited-State Dynamics of Oxyluciferin in Firefly Luciferase.

The color variations of light emitted by some natural and mutant luciferases are normally attributed to collective factors referred to as microenvironment effects; however, the exact nature of these interactions between the emitting molecule (oxyluciferin) and the active site remains elusive. Although model studies of noncomplexed oxyluciferin and its variants have greatly advanced the understanding of its photochemistry, extrapolation of the conclusions to the real system requires assumptions about the polarity and proticity of the active site. To decipher the intricate excited-state dynamics, global and target analysis is performed here for the first time on the steady-state and time-resolved spectra of firefly oxyluciferin complexed with luciferase from the Japanese firefly (Luciola cruciata). The experimental steady-state and time-resolved luminescence spectra of the oxyluciferin/luciferase complex in solution are compared with the broadband time-resolved firefly bioluminescence recorded in vivo. The results demonstrate that de-excitation of the luminophore results in a complex cascade of photoinduced proton transfer processes and can be interpreted by the pH dependence of the emitted light. It is confirmed that proton transfer is the central event in the spectrochemistry of this system for which any assignment of the pH-dependent emission to a single chemical species would be an oversimplification.

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.

Complete Proton Transfer Cycle in GFP and Its T203V and S205V Mutants.

Proton transfer is critical in many important biochemical reactions. The unique three-step excited-state proton transfer in avGFP allows observations of protein proton transport in real-time. In this work we exploit femtosecond to microsecond transient IR spectroscopy to record, in D2 O, the complete proton transfer photocycle of avGFP, and two mutants (T203V and S205V) which modify the structure of the proton wire. Striking differences and similarities are observed among the three mutants yielding novel information on proton transfer mechanism, rates, isotope effects, H-bond strength and proton wire stability. These data provide a detailed picture of the dynamics of long-range proton transfer in a protein against which calculations may be compared.

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.

FRET-FLIM applications in plant systems.

A hallmark of cellular processes is the spatio-temporally regulated interplay of biochemical components. Assessing spatial information of molecular interactions within living cells is difficult using traditional biochemical methods. Developments in green fluorescent protein technology in combination with advances in fluorescence microscopy have revolutionised this field of research by providing the genetic tools to investigate the spatio-temporal dynamics of biomolecules in live cells. In particular, fluorescence lifetime imaging microscopy (FLIM) has become an inevitable technique for spatially resolving cellular processes and physical interactions of cellular components in real time based on the detection of Förster resonance energy transfer (FRET). In this review, we provide a theoretical background of FLIM as well as FRET-FLIM analysis. Furthermore, we show two cases in which advanced microscopy applications revealed many new insights of cellular processes in living plant cells as well as in whole plants.