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Perylene diimide (PDI) dimers and higher aggregates are key components in organic molecular photonics and photovoltaic devices, supporting singlet fission and symmetry breaking charge separation. Detailed understanding of their excited states is thus important. This has proven challenging because interchromophoric coupling is a strong function of dimer architecture. Recently, a macrocyclic PDI dimer was reported in which excitonic coupling could be turned on and off simply by changing the solvent. This presents a useful case where coupling is modified without synthetic changes to tune supramolecular structure. Here we present a detailed study of solvent dependent excited state dynamics in this dimer by means of coherent multidimensional spectroscopy. Spectral analysis resolves the different coupling strengths, which are consistent with solvent dependent changes in dimer conformation. The strongly coupled conformer forms an excimer within 300â fs. The low-frequency Raman active modes recovered from two-dimensional electronic spectra reveal frequencies characteristic of exciton coupling. These are assigned to modes modulating the coupling from the corresponding DFT calculations. Further analysis reveals a time dependent frequency during excimer formation. Analysis of two-dimensional "beatmaps" reveals features in the coupled dimer which are not predicted by the displaced harmonic oscillator model and are assigned to vibronic coupling.
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Unidirectional photochemical molecular motors can act as a power source for molecular machines. The motors operate by successive excited state isomerization and ground state helix inversion reactions, attaining unidirectionality from an interplay of steric strain and stereochemistry. Optimizing the yield of the excited state isomerization reaction is an important goal that requires detailed knowledge of excited state dynamics. Here, we investigate the effect of electron withdrawing and donating substituents on excited state structure and ultrafast dynamics in a series of newly synthesized first generation photochemical molecular motors. All substituents red-shift the absorption spectra, while some modify the Stokes shift and render the fluorescence quantum yield solvent polarity dependent. Raman spectra and density functional theory calculations reveal that the stretching mode of the C=C "axle" in the electronic ground state shows a small red-shift when conjugated with electron withdrawing substituents. Ultrafast fluorescence measurements reveal substituent and solvent polarity effects, with the excited state decay being accelerated by both polar solvent environment and electron withdrawing substituents. Excited state structural dynamics are investigated by fluorescence coherence spectroscopy and femtosecond stimulated Raman spectroscopy. The time resolved Raman measurements are shown to provide structural data specifically on the Franck-Condon excited state. The C=C localized modes have a different substituent dependence compared to the ground state, with the unsubstituted motor having the most red-shifted mode. Such measurements provide valuable new insights into pathways to optimize photochemical molecular motor performance, especially if they can be coupled with high-quality quantum molecular dynamics calculations.
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Vibrational coherences in ultrafast pump-probe (PP) and 2D electronic spectroscopy (2DES) provide insights into the excited state dynamics of molecules. Femtosecond coherence spectra and 2D beat maps yield information about displacements of excited state surfaces for key vibrational modes. Half-broadband 2DES uses a PP configuration with a white light continuum probe to extend the detection range and resolve vibrational coherences in the excited state absorption (ESA). However, the interpretation of these spectra is difficult as they are strongly dependent on the spectrum of the pump laser and the relative displacement of the excited states along the vibrational coordinates. We demonstrate the impact of these convoluting factors for a model based upon cresyl violet. A careful consideration of the position of the pump spectrum can be a powerful tool in resolving the ESA coherences to gain insights into excited state displacements. This paper also highlights the need for caution in considering the spectral window of the pulse when interpreting these spectra.
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Unidirectional photochemically driven molecular motors (PMMs) convert the energy of absorbed light into continuous rotational motion. As such they are key components in the design of molecular machines. The prototypical and most widely employed class of PMMs is the overcrowded alkenes, where rotational motion is driven by successive photoisomerization and thermal helix inversion steps. The efficiency of such PMMs depends upon the speed of rotation, determined by the rate of ground state thermal helix inversion, and the quantum yield of photoisomerization, which is dependent on the excited state energy landscape. The former has been optimized by synthetic modification across three generations of overcrowded alkene PMMs. These improvements have often been at the expense of photoisomerization yield, where there remains room for improvement. In this perspective we review the application of ultrafast spectroscopy to characterize the excited state dynamics in PMMs. These measurements lead to a general mechanism for all generations of PMMs, involving subpicosecond decay of a Franck-Condon excited state to populate a dark excited state which decays within picoseconds via conical intersections with the electronic ground state. The model is discussed in the context of excited state dynamics calculations. Studies of PMM photochemical dynamics as a function of solvent suggest exploitation of intramolecular charge transfer and solvent polarity as a route to controlling photoisomerization yield. A test of these ideas for a first generation motor reveals a high degree of solvent control over isomerization yield. These results suggest a pathway to fine control over the performance of future PMMs.
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Knowledge of relative displacements between potential energy surfaces (PES) is critical in spectroscopy and photochemistry. Information on displacements is encoded in vibrational coherences. Here we apply ultrafast two-dimensional electronic spectroscopy in a pump-probe half-broadband (HB2DES) geometry to probe the ground- and excited-state potential landscapes of cresyl violet. 2D coherence maps reveal that while the coherence amplitude of the dominant 585 cm-1 Raman-active mode is mainly localized in the ground-state bleach and stimulated emission regions, a 338 cm-1 mode is enhanced in excited-state absorption. Modeling these data with a three-level displaced harmonic oscillator model using the hierarchical equation of motion-phase matching approach (HEOM-PMA) shows that the S1 â S0 PES displacement is greater along the 585 cm-1 coordinate than the 338 cm-1 coordinate, while Sn â S1 displacements are similar along both coordinates. HB2DES is thus a powerful tool for exploiting nuclear wavepackets to extract quantitative multidimensional, vibrational coordinate information across multiple PESs.
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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 C2âO 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 C2âO 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 â¼25. Collectively, these studies shed light on the role of the hydrogen bonding network in the LOV ß-scaffold in signal transduction.
Assuntos
Proteínas de Bactérias , Fotorreceptores Microbianos , Proteínas de Bactérias/metabolismo , Análise Espectral , Fotorreceptores Microbianos/química , Bacillus subtilis/metabolismo , Mononucleotídeo de Flavina/metabolismoRESUMO
Photoactivated adenylate cyclases (PACs) are light-activated enzymes that combine a BLUF (blue-light using flavin) domain and an adenylate cyclase domain that are able to increase the levels of the important second messenger cAMP (cyclic adenosine monophosphate) upon blue-light excitation. The light-induced changes in the BLUF domain are transduced to the adenylate cyclase domain via a mechanism that has not yet been established. One critical residue in the photoactivation mechanism of BLUF domains, present in the vicinity of the flavin is the glutamine amino acid close to the N5 of the flavin. The role of this residue has been investigated extensively both experimentally and theoretically. However, its role in the activity of the photoactivated adenylate cyclase, OaPAC has never been addressed. In this work, we applied ultrafast transient visible and infrared spectroscopies to study the photochemistry of the Q48E OaPAC mutant. This mutation altered the primary electron transfer process and switched the enzyme into a permanent 'on' state, able to increase the cAMP levels under dark conditions compared to the cAMP levels of the dark-adapted state of the wild-type OaPAC. Differential scanning calorimetry measurements point to a less compact structure for the Q48E OaPAC mutant. The ensemble of these findings provide insight into the important elements in PACs and how their fine tuning may help in the design of optogenetic devices.
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Adenilil Ciclases , Proteínas de Bactérias , Glutamina , Oscillatoria , Adenilil Ciclases/química , Adenilil Ciclases/genética , Adenilil Ciclases/efeitos da radiação , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/efeitos da radiação , Flavinas/química , Flavinas/efeitos da radiação , Luz , Mutação , Glutamina/genética , Domínios Proteicos/efeitos dos fármacos , Transporte de Elétrons , Ativação Enzimática/efeitos da radiação , Oscillatoria/enzimologiaRESUMO
Two-dimensional electronic spectroscopy (2DES) provides detailed insight into coherent ultrafast molecular dynamics in the condensed phase. Here we report a referenced broadband pump-compressed continuum probe half-broadband (HB) 2DES spectrometer in a partially collinear geometry. To optimize signal-to-noise ratio (SNR) we implement active noise reduction referencing, which has not previously been applied in 2DES. The method is calibrated against the well characterized 2DES response of the oxazine dye cresyl violet and demonstrated at visible wavelengths on the photochromic photoswitch 1,2-Bis(2-methyl-5-phenyl-3-thienyl) perfluorocyclopentene (DAE). The SNR is improved by a factor of â¼2 through active referencing. This is illustrated in an application to resolve a low frequency mode in the excited electronic state of DAE, yielding new data on the reaction coordinate. We show that the active noise reduction referencing, coupled with the rapid data collection, allows the extraction of weak vibronic features, most notably a low frequency mode in the excited electronic state of DAE.
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A significant number of quadrupolar dyes behave as their dipolar analogues when photoexcited in polar environments. This is due to the occurrence of excited-state symmetry breaking (ES-SB), upon which the electronic excitation, initially distributed over the whole molecule, localises preferentially on one side. Here, we investigate the ES-SB properties of two A-D-A dyes, consisting of a pyrrolo-pyrrole donor (D) and either cyanophenyl or dicyanovinyl acceptors (A). For this, we use time-resolved vibrational spectroscopy, comparing IR absorption and femtosecond stimulated Raman spectroscopies. Although dicyanovinyl is a stronger electron-withdrawing group, ES-SB is not observed with the dicyanovinyl-based dye even in highly polar media, whereas it already takes place in weakly polar solvents with dyes containing cyanophenyl accepting groups. This difference is attributed to the large electronic coupling between the D-A branches in the former dye, whose loss upon symmetry breaking cannot be counterbalanced by a gain in solvation energy. Comparison with analogues of the cyanophenyl-based dye containing different spacers reveals that interbranch coupling does not so much depend on the distance between the D-A subunits than on the nature of the spacer. We show that transient Raman spectra probe different modes of these centrosymmetric molecules but are consistent with the transient IR data. However, lifetime broadening of the Raman bands, probably due to the resonance enhancement, may limit the application of this technique for monitoring ES-SB.
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Molecular motors based on the overcrowded alkene motif convert light energy into unidirectional mechanical motion through an excited state isomerization reaction. The realization of experimental control over conversion efficiency in these molecular motors is an important goal. Here, we combine the synthesis of a novel "push-pull" overcrowded alkene motor with photophysical characterization by steady state and ultrafast time-resolved electronic spectroscopy. We show that tuning of the charge transfer character in the excited state has a dramatic effect on the photoisomerization yield, enhancing it to near unity in nonpolar solvents while largely suppressing it in polar solvents. This behavior is explained through reference to solvent- and substituent-dependent potential energy surfaces and their effect on conical intersections to the ground state. These observations offer new routes to the fine control of motor efficiency and introduce additional degrees of freedom in the synthesis and exploitation of light-driven molecular motors.
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Fluorescent labelling of macromolecular samples, including using the green fluorescent protein (GFP), has revolutionised the field of bioimaging. The ongoing development of fluorescent proteins require a detailed understanding of the photophysics of the biochromophore, and how chemical derivatisation influences the excited state dynamics. Here, we investigate the photophysical properties associated with the S1 state of three alkylated derivatives of the chromophore in GFP, in the gas phase using time-resolved photoelectron imaging, and in water using femtosecond fluorescence upconversion. The gas-phase lifetimes (1.6-10 ps), which are associated with the intrinsic (environment independent) dynamics, are substantially longer than the lifetimes in water (0.06-3 ps), attributed to stabilisation of both twisted intermediate structures and conical intersection seams in the condensed phase. In the gas phase, alkylation on the 3 and 5 positions of the phenyl ring slows the dynamics due to inertial effects, while a 'pre-twist' of the methine bridge through alkylation on the 2 and 6 positions significantly shortens the excited state lifetimes. Formation of a minor, long-lived (â« 40 ps) excited state population in the gas phase is attributed to intersystem crossing to a triplet state, accessed because of a T1/S1 degeneracy in the so-called P-trap potential energy minimum associated with torsion of the single-bond in the bridging unit connecting to the phenoxide ring. A small amount of intersystem crossing is supported through TD-DFT molecular dynamics trajectories and MS-CASPT2 calculations. No such intersystem crossing occurs in water at T = 300 K or in ethanol at T ≈ 77 K, due to a significantly altered potential energy surface and P-trap geometry.
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Corantes , Etanol , Proteínas de Fluorescência Verde , Fluorescência , Teoria da Densidade FuncionalRESUMO
Photoactivated adenylate cyclases (PACs) are light activated enzymes that combine blue light sensing capacity with the ability to convert ATP to cAMP and pyrophosphate (PPi) in a light-dependent manner. In most of the known PACs blue light regulation is provided by a blue light sensing domain using flavin which undergoes a structural reorganization after blue-light absorption. This minor structural change then is translated toward the C-terminal of the protein, inducing a larger conformational change that results in the ATP conversion to cAMP. As cAMP is a key second messenger in numerous signal transduction pathways regulating various cellular functions, PACs are of great interest in optogenetic studies. The optimal optogenetic device must be "silent" in the dark and highly responsive upon light illumination. PAC from Oscillatoria acuminata is a very good candidate as its basal activity is very small in the dark and the conversion rates increase 20-fold upon light illumination. We studied the effect of replacing D67 to N, in the blue light using flavin domain. This mutation was found to accelerate the primary electron transfer process in the photosensing domain of the protein, as has been predicted. Furthermore, it resulted in a longer lived signaling state, which was formed with a lower quantum yield. Our studies show that the overall effects of the D67N mutation lead to a slightly higher conversion of ATP to cAMP, which points in the direction that by fine tuning the kinetic properties more responsive PACs and optogenetic devices can be generated.
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Adenilil Ciclases , Proteínas de Bactérias , Oscillatoria , Trifosfato de Adenosina , Adenilil Ciclases/genética , Adenilil Ciclases/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Flavinas/metabolismo , Luz , Sistemas do Segundo Mensageiro , Oscillatoria/enzimologiaRESUMO
The green fluorescent protein (GFP) drove revolutionary progress in bioimaging. Photoconvertible fluorescent proteins (PCFPs) are an important branch of the FP family, of which Kaede is the prototype. Uniquely, PCFPs can be permanently switched from green to red emitting forms on UV irradiation, facilitating applications in site-specific photolabelling and protein tracking. Optimisation and exploitation of FPs requires understanding of the photophysical and photochemical behaviour of the chromophore. Accordingly, the principal GFP chromophore has been the subject of intense experimental and theoretical investigation. In contrast, the photophysics of the red emitting PCFP chromophore are largely unstudied. Here we present a detailed investigation of the excited-state properties of the Kaede chromophore in solution, utilising steady state measurements, ultrafast time-resolved electronic and vibrational spectroscopies, and electronic structure theory. Its excited state dynamics are very different to those of the parent GFP. Most remarkably, the PCFP chromophore has highly complex wavelength-dependent fluorescence decays and a mean lifetime an order of magnitude longer than the GFP chromophore. Transient electronic and vibrational spectroscopies suggest that these dynamics arise from a range of excited-state conformers that are spectrally and kinetically distinct but chemically similar. These conformers are populated directly by excitation of a broad thermal distribution of ground state structures about a single conformer, suggesting an excited-state potential surface with several minima. Temperature-dependence confirms the existence of barriers on the excited-state surface and reveals the radiationless decay mechanism to be internal conversion. These experimental observations are consistent with a model assuming a simple ground state potential energy surface accessing a complex excited state possessing multiple minima.
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Controlling molecular translation at the nanoscale is a key objective for development of synthetic molecular machines. Recently developed third generation photochemically driven molecular motors (3GMs), comprising pairs of overcrowded alkenes capable of cooperative unidirectional rotation offer the possibility of converting light energy into translational motion. Further development of 3GMs demands detailed understanding of their excited state dynamics. Here we use time-resolved absorption and emission to track population and coherence dynamics in a 3GM. Femtosecond stimulated Raman reveals real-time structural dynamics as the excited state evolves from a Franck-Condon bright-state through weakly-emissive dark-state to the metastable product, yielding new insight into the reaction coordinate. Solvent polarity modifies the photoconversion efficiency suggesting charge transfer character in the dark-state. The enhanced quantum yield correlates with suppression of a low-frequency flapping motion in the excited state. This detailed characterization facilitates development of 3GMs, suggesting exploitation of medium and substituent effects to modulate motor efficiency.
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Symmetry breaking charge separation (SBCS) is central to photochemical energy conversion. The widely studied 9,9-bianthryl (9,9'BA) is the prototype, but the role of bianthryl structure is hardly investigated. Here we investigate excited state structural dynamics in a bianthryl of reduced symmetry, 1,9-bianthryl (1,9'BA), through ultrafast electronic and vibrational spectroscopy. Resonance selective Raman in polar solvents reveals a Franck-Condon state mode that disappears concomitant with the rise of ring breathing modes of radical species. Solvent-dependent dynamics show that CS is driven by solvent orientational motion, as in 9,9'BA. In nonpolar solvents the excited state undergoes multistep structural relaxation, including subpicosecond Franck-Condon state decay and biexponential diffusion-controlled structural evolution to a distorted slightly polar state. These data suggest two possible routes to SBCS; the established solvent driven pathway in rapidly relaxing polar solvents and, in slowly relaxing media, initial intramolecular reorganization to a polar structure which drives solvent orientational relaxation.
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Vibração , Solventes/química , Análise Espectral , Movimento (Física)RESUMO
In photosynthesis, nature exploits the distinctive electronic properties of chromophores arranged in supramolecular rings for efficient light harvesting. Among synthetic supramolecular cyclic structures, porphyrin nanorings have attracted considerable attention as they have a resemblance to naturally occurring light-harvesting structures but offer the ability to control ring size and the level of disorder. Here, broadband femtosecond transient absorption spectroscopy, with pump pulses in resonance with either the high or the low energy sides of the inhomogeneously broadened absorption spectrum, is used to study the population dynamics and ground and excited state vibrational coherence in large porphyrin nanorings. A series of fully conjugated, alkyne bridged, nanorings constituted of between ten and forty porphyrin units is studied. Pump-wavelength dependent fast spectral evolution is found. A fast rise or decay of the stimulated emission is found when large porphyrin nanorings are excited on, respectively, the high or low energy side of the absorption spectrum. Such dynamics are consistent with the hypothesis of a variation in transition dipole moment across the inhomogeneously broadened ground state ensemble. The observed dynamics indicate the interplay of nanoring conformation and oscillator strength. Oscillatory dynamics on the sub-ps time domain are observed in both pumping conditions. A combined analysis of the excitation wavelength-dependent transient spectra along with the amplitude and phase evolution of the oscillations allows assignment to vibrational wavepackets evolving on either ground or excited states electronic potential energy surfaces. Even though porphyrin nanorings support highly delocalized electronic wavefunctions, with coherence length spanning tens of chromophores, the measured vibrational coherences remain localised on the monomers. The main contributions to the beatings are assigned to two vibrational modes localised on the porphyrin cores: a Zn-N stretching mode and a skeletal methinic/pyrrolic C-C stretching and in-plane bending mode.
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The hydrogen bonding network that surrounds the flavin in blue light using flavin adenine dinucleotide (BLUF) photoreceptors plays a crucial role in sensing and communicating the changes in the electronic structure of the flavin to the protein matrix upon light absorption. Using time-resolved infrared spectroscopy (TRIR) and unnatural amino acid incorporation, we investigated the photoactivation mechanism and the role of the conserved tyrosine (Y6) in the forward reaction of the photoactivated adenylyl cyclase from Oscillatoria acuminata (OaPAC). Our work elucidates the direct connection between BLUF photoactivation and the structural and functional implications on the partner protein for the first time. The TRIR results demonstrate the formation of the neutral flavin radical as an intermediate species on the photoactivation pathway which decays to form the signaling state. Using fluorotyrosine analogues to modulate the physical properties of Y6, the TRIR data reveal that a change in the pKa and/or reduction potential of Y6 has a profound effect on the forward reaction, consistent with a mechanism involving proton transfer or proton-coupled electron transfer from Y6 to the electronically excited FAD. Decreasing the pKa from 9.9 to <7.2 and/or increasing the reduction potential by 200 mV of Y6 prevents proton transfer to the flavin and halts the photocycle at FADâ¢-. The lack of protonation of the anionic flavin radical can be directly linked to photoactivation of the adenylyl cyclase (AC) domain. While the 3F-Y6 and 2,3-F2Y6 variants undergo the complete photocycle and catalyze the conversion of ATP into cAMP, enzyme activity is abolished in the 3,5-F2Y6 and 2,3,5-F3Y6 variants where the photocycle is halted at FADâ¢-. Our results thus show that proton transfer plays an essential role in initiating the structural reorganization of the AC domain that results in AC activity.
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Adenilil Ciclases , Flavina-Adenina Dinucleotídeo , Trifosfato de Adenosina , Adenilil Ciclases/genética , Aminoácidos , Proteínas de Bactérias/metabolismo , Flavina-Adenina Dinucleotídeo/química , Flavinas/química , Luz , Mutagênese , Prótons , Análise Espectral , TirosinaRESUMO
The photophysics of green fluorescent protein (GFP) and red Kaede fluorescent protein (rKFP) are defined by the intrinsic properties of the light-absorbing chromophore and its interaction with the protein binding pocket. This work deploys photodissociation action spectroscopy to probe the absorption profiles for a series of synthetic GFP and rKFP chromophores as the bare anions and as complexes with the betaine zwitterion, which is assumed as a model for dipole microsolvation. Electronic structure calculations and energy decomposition analysis using Symmetry-Adapted Perturbation Theory are used to characterize gas-phase structures and complex cohesion forces. The calculations reveal a preponderance for coordination of betaine to the phenoxide deprotonation site predominantly through electrostatic forces. Calculations using the STEOM-DLPNO-CCSD method are able to reproduce absolute and relative vertical excitation energies for the bare anions and anion-betaine complexes. On the other hand, treatment of the betaine molecule with a point-charge model, in which the charges are computed from some common electron density population analysis schemes, show that just electrostatic and point-charge induction interactions are unable to account for the betaine-induced spectral shift. The present methodology could be applied to investigate cluster forces and optical properties in other gas-phase ion-zwitterion complexes.
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Eletricidade Estática , Ânions/química , Proteínas de Fluorescência Verde/química , Análise EspectralRESUMO
Light activated proteins are at the heart of photobiology and optogenetics, so there is wide interest in understanding the mechanisms coupling optical excitation to protein function. In addition, such light activated proteins provide unique insights into the real-time dynamics of protein function. Using pump-probe spectroscopy, the function of a photoactive protein can be initiated by a sub-100 fs pulse of light, allowing subsequent protein dynamics to be probed from femtoseconds to milliseconds and beyond. Among the most interesting photoactive proteins are the blue light using flavin (BLUF) domain proteins, which regulate the response to light of a wide range of bacterial and some euglenoid processes. The photosensing mechanism of BLUF domains has long been a subject of debate. In contrast to other photoactive proteins, the electronic and nuclear structure of the chromophore (flavin) is the same in dark- and light-adapted states. Thus, the driving force for photoactivity is unclear.To address this question requires real-time observation of both chromophore excited state processes and their effect on the structure and dynamics of the surrounding protein matrix. In this Account we describe how time-resolved infrared (IR) experiments, coupled with chemical biology, provide important new insights into the signaling mechanism of BLUF domains. IR measurements are sensitive to changes in both chromophore electronic structure and protein hydrogen bonding interactions. These contributions are resolved by isotope labeling of the chromophore and protein separately. Further, a degree of control over BLUF photochemistry is achieved through mutagenesis, while unnatural amino acid substitution allows us to both fine-tune the photochemistry and time resolve protein dynamics with spatial resolution.Ultrafast studies of BLUF domains reveal non-single-exponential relaxation of the flavin excited state. That relaxation leads within one nanosecond to the original flavin ground state bound in a modified hydrogen-bonding network, as seen in transient and steady-state IR spectroscopy. The change in H-bond configuration arises from formation of an unusual enol (imine) form of a critical glutamine residue. The dynamics observed, complemented by quantum mechanical calculations, suggest a unique sequential electron then double proton transfer reaction as the driving force, followed by rapid reorganization in the binding site and charge recombination. Importantly, studies of several BLUF domains reveal an unexpected diversity in their dynamics, although the underlying structure appears highly conserved. It is suggested that this diversity reflects structural dynamics in the ground state at standard temperature, leading to a distribution of structures and photochemical outcomes. Time resolved IR measurements were extended to the millisecond regime for one BLUF domain, revealing signaling state formation on the microsecond time scale. The mechanism involves reorganization of a ß-sheet connected to the chromophore binding pocket via a tryptophan residue. The potential of site-specific labeling amino acids with IR labels as a tool for probing protein structural dynamics was demonstrated.In summary, time-resolved IR studies of BLUF domains (along with related studies at visible wavelengths and quantum and molecular dynamics calculations) have resolved the photoactivation mechanism and real-time dynamics of signaling state formation. These measurements provide new insights into protein structural dynamics and will be important in optimizing the potential of BLUF domains in optobiology.
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Proteínas de Bactérias , Flavinas , Proteínas de Bactérias/química , Transporte de Elétrons , Flavinas/química , Ligação de Hidrogênio , Estrutura Terciária de ProteínaRESUMO
RsEGFP2 is a reversibly photoswitchable fluorescent protein used in super-resolved optical microscopies, which can be toggled between a fluorescent On state and a nonfluorescent Off state. Previous time-resolved ultraviolet-visible spectroscopic studies have shown that the Off-to-On photoactivation extends over the femto- to millisecond time scale and involves two picosecond lifetime excited states and four ground state intermediates, reflecting a trans-to-cis excited state isomerization, a millisecond deprotonation, and protein structural reorganizations. Femto- to millisecond time-resolved multiple-probe infrared spectroscopy (TRMPS-IR) can reveal structural aspects of intermediate species. Here we apply TRMPS-IR to rsEGFP2 and implement a Savitzky-Golay derivative analysis to correct for baseline drift. The results reveal that a subpicosecond twisted excited state precursor controls the trans-to-cis isomerization and the chromophore reaches its final position in the protein pocket within 100 ps. A new step with a time constant of 42 ns is reported and assigned to structural relaxation of the protein that occurs prior to the deprotonation of the chromophore on the millisecond time scale.