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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 β-scaffold in signal transduction.

The transport of microplastics through the atmosphere has been acknowledged as a significant route for their dispersion across different environments. Microplastics of fibrous shape often prevail in environmental samples, although their composition identification might be challenging and lead to an overestimation of plastic microfibers (MFs). Conversely, MFs of natural origin are rarely reported in microplastics studies despite the lack of consensus on the risks they may pose to the environment. In this study, airborne MFs collected in a sparsely populated residential area on the shore of the Eastern Red Sea were analyzed to investigate their abundance and polymer composition and assess their potential transport and deposition rates. The length of observed fibers ranged from 183 μm to 11,877 μm, with 3 % of fibers being >5 mm. The average length of MFs (< 5 mm) was 1378 ± 934 μm. Plastic MFs comprised 10 % of all identified MFs, with polyester being the most common plastic polymer (81.25 %). The mean abundance of airborne MFs was 0.9 ± 0.8 × 10 -2 MFs m -3 . The estimated mean atmospheric microfibrous deposition was 70 MFs m -2 d -1 , with a component of 8 plastic MFs m -2 d -1 . Based on the HYSPLIT backward trajectory analysis, fibers of local origin (estimated to travel approximately 25 km before sampling) were deposited at the sampling location. Air masses of northwestern origin traveling along the coast of the Eastern Red Sea dominated, potentially reducing the abundance of airborne MFs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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 ¾ th 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

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.

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.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

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., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

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 AppA BLUF by the introduction of fluorotyrosine (F-Tyr) analogues that modulated the pK a 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 pK a 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 AppA BLUF , modulation in the Tyr (Y8) pK a has a profound impact on the forward photoreaction. In particular, a decrease in Y8 pK a 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 pK a on the rate of dark recovery is markedly reduced in PixD. These observations highlight very significant differences between the photocycles of PixD and AppA BLUF , despite their sharing highly conserved FAD binding architectures.

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.

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 β-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.

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.

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.

The photophysics of the chromophore of the green fluorescent protein in Aequorea victoria (avGFP) are dominated by an excited state proton transfer reaction. In contrast the photophysics of the same chromophore in solution are dominated by radiationless decay, and photoacid behaviour is not observed. Here we show that modification of the p K a of the chromophore by fluorination leads to an excited state proton transfer on an extremely fast (50 fs) time scale. Such a fast rate suggests a barrierless proton transfer and the existence of a pre-formed acceptor site in the aqueous solution, which is supported by solvent and deuterium isotope effects. In addition, at lower pH, photochemical formation of the elusive zwitterion of the GFP chromophore is observed by means of an equally fast excited state proton transfer from the cation. The significance of these results for understanding and modifying the properties of fluorescent proteins are discussed.

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., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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 (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.

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.

This chapter describes the procedure for globally analyzing fluorescence lifetime imaging (FLIM) data for the observation and quantification of Förster resonance energy transfer (FRET) in live plant cells. The procedure is illustrated by means of a case study, for which plant protoplasts were transfected with different visible fluorescent proteins and subsequently imaged using two-photon excitation FLIM. Spatially resolved fluorescence lifetime images were obtained by application of global analysis using the program Glotaran, which is open-source and freely available software. Using this procedure it is possible to extract the fraction and distance of interacting species between, or conformational changes within proteins, from complex experimental FRET-FLIM datasets, even at low signal-to-noise ratios. In addition, the software allows excluding inherently present autofluorescence from the plant cells, which improves the accuracy of the FRET analysis. The results from the case study are presented and interpreted in the context of the current scientific understanding of these biological systems.

This chapter describes a procedure of global analysis of the steady-state spectra measured with different concentrations of the denaturant to quantitatively study protein denaturation. With the help of physicochemical models, relevant spectral parameters that characterize the folding intermediate and thermodynamic parameters that describe a three-state model N-I-U can be estimated.

This chapter describes an experimental layout for time and spectrally resolved fluorescence measurements with femtosecond time resolution based on Kerr gating. The combination of data recorded using different Kerr media allows a temporal dynamic range from ~100 fs to several nanoseconds. Simultaneous analysis of multiple datasets is described.

Hef is an archaeal member of the DNA repair endonuclease XPF (XPF)/Crossover junction endonuclease MUS81 (MUS81)/Fanconi anemia, complementation group M (FANCM) protein family that in eukaryotes participates in the restart of stalled DNA replication forks. To investigate the physiological roles of Hef in maintaining genome stability in living archaeal cells, we studied the localization of Hef-green fluorescent protein fusions by fluorescence microscopy. Our studies revealed that Haloferax volcanii Hef proteins formed specific localization foci under regular growth conditions, the number of which specifically increased in response to replication arrest. Purification of the full-length Hef protein from its native host revealed that it forms a stable homodimer in solution, with a peculiar elongated configuration. Altogether our data indicate that the shape of Hef, significant physicochemical constraints and/or interactions with DNA limit the apparent cytosolic diffusion of halophilic DNA replication/repair complexes, and demonstrate that Hef proteins are dynamically recruited to archaeal eukaryotic-like chromatin to counteract DNA replication stress. We suggest that the evolutionary conserved function of Hef/FANCM proteins is to enhance replication fork stability by directly interacting with collapsed replication forks.

In many bacteria the flavoenzyme thymidylate synthase ThyX produces the DNA nucleotide deoxythymidine monophosphate from dUMP, using methylenetetrahydrofolate as carbon donor and NADPH as hydride donor. Because all three substrates bind in close proximity to the catalytic flavin adenine dinucleotide group, substantial flexibility of the ThyX active site has been hypothesized. Using femtosecond time-resolved fluorescence spectroscopy, we have studied the conformational heterogeneity and the conformational interconversion dynamics in real time in ThyX from the hyperthermophilic bacterium Thermotoga maritima. The dynamics of electron transfer to excited flavin adenine dinucleotide from a neighboring tyrosine residue are used as a sensitive probe of the functional dynamics of the active site. The fluorescence decay spanned a full three orders of magnitude, demonstrating a very wide range of conformations. In particular, at physiological temperatures, multiple angstrom cofactor-residue displacements occur on the picoseconds timescale. These experimental findings are supported by molecular dynamics simulations. Binding of the dUMP substrate abolishes this flexibility and stabilizes the active site in a configuration where dUMP closely interacts with the flavin cofactor and very efficiently quenches fluorescence itself. Our results indicate a dynamic selected-fit mechanism where binding of the first substrate dUMP at high temperature stabilizes the enzyme in a configuration favorable for interaction with the second substrate NADPH, and more generally have important implications for the role of active site flexibility in enzymes interacting with multiple poly-atom substrates and products. Moreover, our data provide the basis for exploring the effect of inhibitor molecules on the active site dynamics of ThyX and other multisubstrate flavoenzymes.

The first systematic pico-nanosecond time-resolved spectroscopic study of the firefly emitter oxyluciferin and two of its chemically modified analogues revealed that in the excited state the enol group is more acidic than the phenol group. The 6'-dehydroxylated derivative, in which only the 4-enolic hydroxyl proton is acidic, has an experimentally determined pK(a)* of 0.9 in dimethyl sulfoxide and an estimated pK(a)* of -0.3 in water. Moreover, this compound provided direct evidence that in a nonpolar, basic environment the keto form in the excited state can tautomerize into the enol, which subsequently undergoes excited-state proton transfer (ESPT) to produce enolate ion. This observation presents the first experimental evidence of excited-state keto-enol tautomerization of a firefly fluorophore, and it could be important in resolving the enol-keto conundrum related to the color-tuning mechanism of firefly bioluminescence. The 6'-dehydroxylated form of oxyluciferin adds a very rare case of a stable enol to the family of "super"photoacids.

Pyrococcus abyssi NucS is the founding member of a new family of structure-specific DNA endonucleases that interact with the replication clamp proliferating cell nuclear antigen (PCNA). Using a combination of small angle x-ray scattering and surface plasmon resonance analyses, we demonstrate the formation of a stable complex in solution, in which one molecule of the PabNucS homodimer binds to the outside surface of the PabPCNA homotrimer. Using fluorescent labels, PCNA is shown to increase the binding affinity of NucS toward single-strand/double-strand junctions on 5' and 3' flaps, as well as to modulate the cleavage specificity on the branched DNA structures. Our results indicate that the presence of a single major contact between the PabNucS and PabPCNA proteins, together with the complex-induced DNA bending, facilitate conformational flexibility required for specific cleavage at the single-strand/double-strand DNA junction.

Yellow Cameleon 3.60 (YC3.60) is a calcium sensor based on Förster resonance energy transfer (FRET). This sensor is composed of a calmodulin domain and a M13 peptide, which are located in between enhanced cyan-fluorescent protein (ECFP) and the Venus variant of enhanced yellow-fluorescent protein (EYFP). Depending on the calcium concentration, the efficiency of FRET from donor ECFP to acceptor EYFP is changing. In this study, we have recorded time-resolved fluorescence spectra of ECFP, EYFP, and YC3.60 in aqueous solution with picosecond time resolution, using different excitation wavelengths. Detailed insight in the FRET kinetics was obtained by using global and target analyses of time- and wavelength-resolved fluorescence of purified YC3.60 in calcium-free and calcium-bound conformations. The results clearly demonstrate that for both conformations, there are two distinct donor populations: a major one giving rise to FRET and a minor one not able to perform FRET. The transfer time for the calcium-bound conformation is 21 ps, whereas it is in the order of 1 ns for the calcium-free conformation. Ratio imaging of acceptor and donor fluorescence intensities of YC3.60 is usually applied to measure Ca(2+) concentrations in living cells. From the obtained results, it is clear that the intensity ratio is strongly influenced by the presence of donor molecules that do not take part in FRET, thereby significantly affecting the quantitative interpretation of the results.

During denaturant-induced equilibrium (un)folding of wild-type apoflavodoxin from Azotobacter vinelandii, a molten globule-like folding intermediate is formed. This wild-type protein contains three tryptophans. In this study, we use a general approach to analyze time-resolved fluorescence and steady-state fluorescence data that are obtained upon denaturant-induced unfolding of a single-tryptophan-containing variant of apoflavodoxin [i.e., W74/F128/F167 (WFF) apoflavodoxin]. The experimental data are assembled in matrices, and subsequent singular-value decomposition of these matrices (i.e., based on either steady-state or time-resolved fluorescence data) shows the presence of three significant, and independent, components. Consequently, to further analyze the denaturation trajectories, we use a three-state protein folding model in which a folding intermediate and native and unfolded protein molecules take part. Using a global analysis procedure, we determine the relative concentrations of the species involved and show that the stability of WFF apoflavodoxin against global unfolding is ∼4.1 kcal/mol. Analysis of time-resolved anisotropy data of WFF apoflavodoxin unfolding reveals the remarkable observation that W74 is equally well fixed within both the native protein and the molten globule-like folding intermediate. Slight differences between the direct environments of W74 in the folding intermediate and native protein cause different rotameric populations of the indole in both folding species as fluorescence lifetime analysis reveals. Importantly, thermodynamic analyses of the spectral denaturation trajectories of the double-tryptophan-containing protein variants WWF apoflavodoxin and WFW apoflavodoxin show that these variants are significantly more stable (5.9 kcal/mol and 6.8 kcal/mol, respectively) than WFF apoflavodoxin (4.1 kcal/mol) Hence, tryptophan residues contribute considerably to the 10.5 kcal/mol thermodynamic stability of native wild-type apoflavodoxin.

Recently, we described that ATP induces changes in YFP/CFP fluorescence intensities of Fluorescence Resonance Energy Transfer (FRET) sensors based on CFP-YFP. To get insight into this phenomenon, we employed fluorescence lifetime spectroscopy to analyze the influence of ATP on these fluorescent proteins in more detail. Using different donor and acceptor pairs we found that ATP only affected the CFP-YFP based versions. Subsequent analysis of purified monomers of the used proteins showed that ATP has a direct effect on the fluorescence lifetime properties of CFP. Since the fluorescence lifetime analysis of CFP is rather complicated by the existence of different lifetimes, we tested a variant of CFP, i.e. Cerulean, as a monomer and in our FRET constructs. Surprisingly, this CFP variant shows no ATP concentration dependent changes in the fluorescence lifetime. The most important difference between CFP and Cerulean is a histidine residue at position 148. Indeed, changing this histidine in CFP into an aspartic acid results in identical fluorescence properties as observed for the Cerulean fluorescent based FRET sensor. We therefore conclude that the changes in fluorescence lifetime of CFP are affected specifically by possible electrostatic interactions of the negative charge of ATP with the positively charged histidine at position 148. Clearly, further physicochemical characterization is needed to explain the sensitivity of CFP fluorescence properties to changes in environmental (i.e. ATP concentrations) conditions.

Picosecond fluorescence kinetics of wild-type (WT) and mutant cells of Synechocystis sp. PCC 6803, were studied at the ensemble level with a streak-camera and at the cell level using fluorescence-lifetime-imaging microscopy (FLIM). The FLIM measurements are in good agreement with the ensemble measurements, but they (can) unveil variations between and within cells. The BE mutant cells, devoid of photosystem II (PSII) and of the light-harvesting phycobilisomes, allowed the study of photosystem I (PSI) in vivo for the first time, and the observed 6-ps equilibration process and 25-ps trapping process are the same as found previously for isolated PSI. No major differences are detected between different cells. The PAL mutant cells, devoid of phycobilisomes, show four lifetimes: ∼20 ps (PSI and PSII), ∼80 ps, ∼440 ps, and 2.8 ns (all due to PSII), but not all cells are identical and variations in the kinetics are traced back to differences in the PSI/PSII ratio. Finally, FLIM measurements on WT cells reveal that in some cells or parts of cells, phycobilisomes are disconnected from PSI/PSII. It is argued that the FLIM setup used can become instrumental in unraveling photosynthetic regulation mechanisms in the future., (Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

We investigated the effects of digalactosyl-diacylglycerol (DGDG) on the organization and thermal stability of thylakoid membranes, using wild-type Arabidopsis thaliana and the DGDG-deficient mutant, dgd1. Circular-dichroism measurements reveal that DGDG-deficiency hampers the formation of the chirally organized macrodomains containing the main chlorophyll a/b light-harvesting complexes. The mutation also brings about changes in the overall chlorophyll fluorescence lifetimes, measured in whole leaves as well as in isolated thylakoids. As shown by time-resolved measurements, using the lipophylic fluorescence probe Merocyanine 540 (MC540), the altered lipid composition affects the packing of lipids in the thylakoid membranes but, as revealed by flash-induced electrochromic absorbance changes, the membranes retain their ability for energization. Thermal stability measurements revealed more significant differences. The disassembly of the chiral macrodomains around 55°C, the thermal destabilization of photosystem I complex at 61°C as detected by green gel electrophoresis, as well as the sharp drop in the overall chlorophyll fluorescence lifetime above 45°C (values for the wild type-WT) occur at 4-7°C lower temperatures in dgd1. Similar differences are revealed in the temperature dependence of the lipid packing and the membrane permeability: at elevated temperatures MC540 appears to be extruded from the dgd1 membrane bilayer around 35°C, whereas in WT, it remains lipid-bound up to 45°C and dgd1 and WT membranes become leaky around 35 and 45°C, respectively. It is concluded that DGDG plays important roles in the overall organization of thylakoid membranes especially at elevated temperatures.

A methodology is described for the quantitative determination of Förster resonance energy transfer (FRET) in live cells using the rise time of acceptor fluorescence as determined with fluorescence lifetime imaging microscopy (FLIM). An advantage of this method is that only those molecules that are involved in the energy-transfer process are monitored. This contrasts with current methods that measure either steady-state fluorescence of donor and acceptor molecules or time-resolved fluorescence of donor molecules, and thereby probe a mixture of donor molecules that are involved in FRET and those that are fluorescent but not involved in FRET. The absence of FRET can, for instance, be due to unwanted acceptor bleaching or incomplete maturing of visible proteins that should act as acceptor molecules. In addition, parameters describing the rise of acceptor fluorescence and the decay of donor fluorescence can be determined via simultaneous global analysis of multiple FLIM images, thereby increasing the reliability of the analysis. In the present study, plant protoplasts transfected with fusions of visible fluorescent proteins are used to illustrate the new data analysis method. It is demonstrated that the distances estimated with the present method are substantially smaller than those estimated from the average donor lifetimes, due to a fraction of non-transferring donor molecules. Software to reproduce the presented results is provided in an open-source and freely available package called "TIMP" for "The R project for Statistical Computing".

A high-throughput Förster resonance energy transfer (FRET) study was performed on the approximately 100 amino acids long N-terminal domain of the photosynthetic complex CP29 of higher plants. For this purpose, CP29 was singly mutated along its N-terminal domain, replacing one-by-one native amino acids by a cysteine, which was labeled with a BODIPY fluorescent probe, and reconstituted with the natural pigments of CP9, chlorophylls and xanthophylls. Picosecond fluorescence experiments revealed rapid energy transfer (approximately 20-70 ps) from BODIPY at amino-acid positions 4, 22, 33, 40, 56, 65, 74, 90, and 97 to Chl a molecules in the hydrophobic part of the protein. From the energy transfer times, distances were estimated between label and chlorophyll molecules, using the Förster equation. When the label was attached to amino acids 4, 56, and 97, it was found to be located very close to the protein core (approximately 15 A), whereas labels at positions 15, 22, 33, 40, 65, 74, and 90 were found at somewhat larger distances. It is concluded that the entire N-terminal domain is in close contact with the hydrophobic core and that there is no loop sticking out into the stroma. Most of the results support a recently proposed topological model for the N-terminus of CP29, which was based on electron-spin-resonance measurements on spin-labeled CP29 with and without its natural pigment content. The present results lead to a slight refinement of that model.

Förster resonance energy transfer (FRET) is a powerful method for obtaining information about small-scale lengths between biomacromolecules. Visible fluorescent proteins (VFPs) are widely used as spectrally different FRET pairs, where one VFP acts as a donor and another VFP as an acceptor. The VFPs are usually fused to the proteins of interest, and this fusion product is genetically encoded in cells. FRET between VFPs can be determined by analysis of either the fluorescence decay properties of the donor molecule or the rise time of acceptor fluorescence. Time-resolved fluorescence spectroscopy is the technique of choice to perform these measurements. FRET can be measured not only in solution, but also in living cells by the technique of fluorescence lifetime imaging microscopy (FLIM), where fluorescence lifetimes are determined with the spatial resolution of an optical microscope. Here we focus attention on time-resolved fluorescence spectroscopy of purified, selected VFPs (both single VFPs and FRET pairs of VFPs) in cuvette-type experiments. For quantitative interpretation of FRET-FLIM experiments in cellular systems, details of the molecular fluorescence are needed that can be obtained from experiments with isolated VFPs. For analysis of the time-resolved fluorescence experiments of VFPs, we have utilised the maximum entropy method procedure to obtain a distribution of fluorescence lifetimes. Distributed lifetime patterns turn out to have diagnostic value, for instance, in observing populations of VFP pairs that are FRET-inactive.

Addition of calcium ions to the Ca(2+)-regulated photoproteins, such as aequorin and obelin, produces a blue bioluminescence originating from a fluorescence transition of the protein-bound product, coelenteramide. The kinetics of several transient fluorescent species of the bound coelenteramide is resolved after picosecond-laser excitation and streak camera detection. The initially formed spectral distributions at picosecond-times are broad, evidently comprised of two contributions, one at higher energy (approximately 25,000 cm(-1)) assigned as from the Ca(2+)-discharged photoprotein-bound coelenteramide in its neutral state. This component decays much more rapidly (t(1/2) approximately 2 ps) in the case of the Ca(2+)-discharged obelin than aequorin (t(1/2) approximately 30 ps). The second component at lower energy shows several intermediates in the 150-500 ps times, with a final species having spectral maxima 19 400 cm(-1), bound to Ca(2+)-discharged obelin, and 21 300 cm(-1), bound to Ca(2+)-discharged aequorin, and both have a fluorescence decay lifetime of 4 ns. It is proposed that the rapid kinetics of these fluorescence transients on the picosecond time scale, correspond to times for relaxation of the protein structural environment of the binding cavity.

Förster resonance energy transfer (FRET) is a widely used method for monitoring interactions between or within biological macromolecules conjugated with suitable donor-acceptor pairs. Donor fluorescence lifetimes in absence and presence of acceptor molecules are often measured for the observation of FRET. However, these lifetimes may originate from interacting and noninteracting molecules, which hampers quantitative interpretation of FRET data. We describe a methodology for the detection of FRET that monitors the rise time of acceptor fluorescence on donor excitation thereby detecting only those molecules undergoing FRET. The large advantage of this method, as compared to donor fluorescence quenching method used more commonly, is that the transfer rate of FRET can be determined accurately even in cases where the FRET efficiencies approach 100% yielding highly quenched donor fluorescence. Subsequently, the relative orientation between donor and acceptor chromophores is obtained from time-dependent fluorescence anisotropy measurements carried out under identical conditions of donor excitation and acceptor detection. The FRET based calcium sensor Yellow Cameleon 3.60 (YC3.60) was used because it changes its conformation on calcium binding, thereby increasing the FRET efficiency. After mapping distances and orientation angles between the FRET moieties in YC3.60, cartoon models of this FRET sensor with and without calcium could be created. Independent support for these representations came from experiments where the hydrodynamic properties of YC3.60 under ensemble and single-molecule conditions on selective excitation of the acceptor were determined. From rotational diffusion times as found by fluorescence correlation spectroscopy and consistently by fluorescence anisotropy decay analysis it could be concluded that the open structure (without calcium) is flexible as opposed to the rather rigid closed conformation. The combination of two independent methods gives consistent results and presents a rapid and specific methodology to analyze structural and dynamical changes in a protein on ligand binding.