Unraveling Ultrafast Photoinduced Proton Transfer Dynamics in a Fluorescent Protein Biosensor for Ca2+ Imaging

Imaging Ca²⁺ dynamics in living systems holds great potential to advance neuroscience and cellular biology. G‐GECO1.1 is an intensiometric fluorescent protein Ca²⁺ biosensor with a Thr‐Tyr‐Gly chromophore. The protonated chromophore emits green upon photoexcitation via excited‐state proton transfer (ESPT). Upon Ca²⁺ binding, a significant population of the chromophores becomes deprotonated. It remains elusive how the chromophore structurally evolves prior to and during ESPT, and how it is affected by Ca²⁺. We use femtosecond stimulated Raman spectroscopy to dissect ESPT in both the Ca²⁺‐free and bound states. The protein chromophores exhibit a sub‐200 fs vibrational frequency shift due to coherent small‐scale proton motions. After wavepackets move out of the Franck–Condon region, ESPT gets faster in the Ca²⁺‐bound protein, indicative of the formation of a more hydrophilic environment. These results reveal the governing structure–function relationship of Ca²⁺‐sensing protein biosensors.     

Role of Ser65, His148 and Thr203 in the Organic Solvent‐dependent Spectral Shift in Green Fluorescent Protein

The photophysics of green fluorescent protein (GFP) is remarkable because of its exceptional property of excited state proton transfer (ESPT) and the presence of a functional proton wire. Another interesting property of wild‐type GFP is that its absorption and fluorescence excitation spectra are sensitive to the presence of polar organic solvents even at very low concentrations. Here, we use a combination of methodologies including site‐specific mutagenesis, absorption spectroscopy, steady‐state and time‐resolved fluorescence measurements and all‐atom molecular dynamics simulations in explicit solvent, to uncover the mechanism behind the unique spectral sensitivity of GFP toward organic solvents. Based on the evidences provided herein, we suggest that organic solvent‐induced changes in the proton wire prevent ground state movement of a proton through the wire and thus bring about the spectral changes observed. The present study can not only help to understand the mechanism of proton transfer by further dissecting the intricate steps in GFP photophysics but also encourages to develop GFP‐based organic solvent biosensors.     

Excited-state dynamics of phenol-pyridinium biaryl

The excited-state dynamics of a donor-acceptor phenol-pyridinium biaryl cation was investigated in various solvents by femtosecond transient absorption spectroscopy and temperature dependent steady-state emission measurements. After excitation to a near-planar Franck-Condon delocalized excited S(1)(DE) state with mesomeric character, three fast relaxation processes are well resolved: solvation, intramolecular rearrangement leading to a twisted charge-shift (CSh) S(1) state with localized character, and excited-state proton transfer (ESPT) to the solvent leading to the phenoxide-pyridinium zwitterion. The proton transfer kinetics depends on the proton accepting character of the solvent whereas the interring torsional kinetics depends on the solvent polarity and viscosity. In nitriles, ESPT does not occur and interring twisting arises with no significant intrinsic barrier, but still slower than solvation. The CSh state is notably fluorescent. In alcohols and water, ESPT is faster than the solvation and DE → CSh relaxation processes and yields the zwitterion hot ground state, which strongly quenches the fluorescence. In THF, solvation and interring twisting occur first, leading to the fully relaxed, weakly fluorescent CSh state, followed by slow ESPT towards the zwitterion. At low temperature (77 K), the large viscous barrier of the solvent inhibits the torsional relaxation but ESPT still arises to some extent. Strong emission from the DE geometry and planar zwitterion is thus observed. Finally, quantum chemical calculations were performed on the ground and excited state of model phenol-pyridinium and phenoxide-pyridinium compounds. Strong S(1) state energy stabilization is predicted upon twisting in both cases, consistent with a fast relaxation towards the perpendicular geometry. A substantial S(0)-S(1) energy gap is still present for the twisted cationic species, which can explain the long-lived emission of the CSh state in nitriles. A quite different situation arises with the zwitterion for which the S(0)-S(1) energy gap predicted at the twisted geometry is very small. This suggests a close-lying conical intersection and can account for the strong fluorescence quenching observed in solvents where the zwitterion is produced by ESPT.     

Competition between energy and proton transfer in ultrafast excited-state dynamics of an oligomeric fluorescent protein red Kaede

We investigated femtosecond and picosecond time-resolved fluorescence dynamics of a tetrameric fluorescent protein Kaede with a red chromophore (red Kaede) to examine a relationship between the excited-state dynamics and a quaternary structure of the fluorescent protein. Red Kaede was obtained by photoconversion from green Kaede that was cloned from a stony coral Trachyphyllia geoffroyi. In common with other typical fluorescent proteins, a chromophore of red Kaede has two protonation states, the neutral and the anionic forms in equilibrium. Time-resolved fluorescence measurements clarified that excitation of the neutral form gives the anionic excited state with a time constant of 13 ps at pH 7.5. This conversion process was attributed to fluorescence resonance energy transfer (FRET) from the photoexcited neutral form to the ground-state anionic form that is located in an adjacent subunit in the tetramer. The time-resolved fluorescence data measured at different pH revealed that excited-state proton transfer (ESPT) also occurs with a time constant of 300 ps and hence that the FRET and ESPT take place simultaneously in the fluorescent protein as competing processes. The ESPT rate in red Kaede was significantly slower than the rate in Aequorea GFP, which highly likely arises from the different hydrogen bond network around the chromophore.     

Controlling Light-Induced Proton Transfer from the GFP Chromophore

Green Fluorescent Protein (GFP) is known to undergo excited-state proton transfer (ESPT). Formation of a short H-bond favors ultrafast ESPT in GFP-like proteins, such as the GFP S65T/H148D mutant, but the detailed mechanism and its quantum nature remain to be resolved. Here we study in vacuo, light-induced proton transfer from the GFP chromophore in hydrogen-bonded complexes with two anionic proton acceptors, I⁻ and deprotonated trichloroacetic acid (TCA⁻). We address the role of the strong H-bond and the quantum mechanical proton-density distribution in the excited state, which determines the proton-transfer probability. Our study shows that chemical modifications to the molecular network drastically change the proton-transfer probability and it can become strongly wavelength dependent. The proton-transfer branching ratio is found to be 60 % for the TCA complex and 10 % for the iodide complex, being highly dependent on the photon energy in the latter case. Using high-level ab initio calculations, we show that light-induced proton transfer takes place in S₁, revealing intrinsic photoacid properties of the isolated GFP chromophore in strongly bound H-bonded complexes. ESPT is found to be very sensitive to the topography of the highly anharmonic potential in S₁, depending on the quantum-density distribution upon vibrational excitation. We also show that the S₁ potential-energy surface, and hence excited-state proton transfer, can be controlled by altering the chromophore microenvironment.     

The effect of hydrogen bonding on the excited-state proton transfer in 2-(2'-hydroxyphenyl)benzothiazole: a TDDFT molecular dynamics study

The dynamics of the excited-state proton transfer (ESPT) in a cluster of 2-(2'-hydroxyphenyl)benzothiazole (HBT) and hydrogen-bonded water molecules was investigated by means of quantum chemical simulations. Two different enol ground-state structures of HBT interacting with the water cluster were chosen as initial structures for the excited-state dynamics: (i) an intramolecular hydrogen-bonded structure of HBT and (ii) a cluster where the intramolecular hydrogen bond in HBT is broken by intermolecular interactions with water molecules. On-the-fly dynamics simulations using time-dependent density functional theory show that after photoexcitation to the S(1) state the ESPT pathway leading to the keto form strongly depends on the initial ground state structure of the HBT-water cluster. In the intramolecular hydrogen-bonded structures direct excited-state proton transfer is observed within 18 fs, which is a factor two faster than proton transfer in HBT computed for the gas phase. Intermolecular bonded HBT complexes show a complex pattern of excited-state proton transfer involving several distinct mechanisms. In the main process the tautomerization proceeds via a triple proton transfer through the water network with an average proton transfer time of approximately 120 fs. Due to the lack of the stabilizing hydrogen bond, intermolecular hydrogen-bonded structures have a significant degree of interring twisting already in the ground state. During the excited state dynamics, the twist tends to quickly increase indicating that internal conversion to the electronic ground state should take place at the sub-picosecond scale.     

Spectroscopy and femtosecond dynamics of excited-state proton transfer induced charge transfer reaction

Fluorescence spectroscopy and femtosecond relaxation dynamics of 2-{[2-(2-hydroxyphenyl)benzo[d]oxazol-6-yl]methylene}malononitrile (diCN-HBO) and 2-{[2-(2-hydroxyphenyl)benzo[d]thiazol-6-yl]methylene}malononitrile (diCN-HBT) are studied to probe the excited-state proton transfer (ESPT) coupled charge transfer (ESCT) reaction. Unlike most of the ESPT/ESCT systems previously designed, in which ESCT takes place prior to ESPT, both diCN-HBO and diCN-HBT undergo ESPT, concomitantly accompanied with the charge transfer process, such that the ESPT reaction dynamics are directly coupled with solvent polarization effects. The long-range solvent polarization interactions result in a solvent-induced barrier that affects the overall proton transfer reaction rate. In cyclohexane, the rate constant of ESPT of diCN-HBO is measured to be 1.1 ps (9.1 x 10(11) s(-1)), which is apparently slower than that of 150 fs for the parent molecule 2-(2'-hydroxyphenyl)benzoxazole (HBO). Upon increasing solvent polarity to, for example, CH 3CN, the rate of ESPT is increased to 300 fs (3.3 x 10(12) s(-1)). The results are rationalized by the stabilization of proton transfer tautomer, which possesses a large degree of charge transfer character via an increase of the solvent polarity, such that the corresponding solvent-induced barrier is reduced. We thus demonstrate a prototypical system in which the photon-induced nuclear motion (proton transfer) is directly coupled with solvent polarization and the corresponding mechanism is reminiscent of that applied in an electron transfer process.     

Proton translocation in monomolecular Langmuir-Blodgett films including 2-naphthol and 1,4-anthraquinone derivatives

The scope of the present work is the investigation of proton transport through monomolecular Langmuir-Blodgett (LB) films. The films were formed from amphiphilic molecules: 2-naphtholo-6-sulfonamide of dodecylamine (N) and 1,4-anthraquinono-2 sulfonamide of dodecylamine (A). The 2-naphthol derivative can act as a proton donor due to excited state proton transfer (ESPT) and the 1,4-anthraquinone group can play the role of proton acceptor because of protonation of the reduced form if it is present. Absorption and emission spectra of LB films containing N and A were registered and separated into component bands. Individual absorption and emission peaks observed were assigned to given forms of chromophores. The behavior of different component bands reflects the state of anthraquinone dependent on proton concentration. A correlation of rate and efficiency of ESPT, with changes of the spectra of A, may be expected to yield information concerning the transport of protons from N to A. The influence of the donor-acceptor distance, sample humidity, film arrangement and the presence of protonophores (Gramicidin A) on proton transfer is studied. Our results indicate that the proton can be transported through the film but its concentration vanishes at the distance greater than 30 A. The efficiency of proton transfer depends strongly on water content, film structure and the presence of ion channels.     

Role of solvation dynamics in excited state proton transfer of 1-naphthol in nanoscopic water clusters formed in a hydrophobic solvent

Excited state proton transfer (ESPT) in biologically relevant organic molecules in aqueous environments following photoexcitation is very crucial as the reorganization of polar solvents (solvation) in the locally excited (LE) state of the organic molecule plays an important role in the overall rate of the ESPT process. A clear evolution of the two photoinduced dynamics in a model ESPT probe 1-naphthol (NpOH) upon ultrafast photoexcitation is the motive of the present study. Herein, the detailed kinetics of the ESPT reaction of NpOH in water clusters formed in hydrophobic solvent are investigated. Distinct values of time constants associated with proton transfer and solvent relaxation have been achieved through picosecond-resolved fluorescence measurements. We have also used a model solvation probe Coumarin 500 (C500) to investigate the dynamics of solvation in the same environmental condition. The temperature dependent picosecond-resolved measurement of ESPT of NpOH and the dynamics of solvation from C500 identify the magnitude of intermolecular hydrogen bonding energy in the water cluster associated with the ultrafast ESPT process.     

Dynamics and prototropic reactivity of electronically excited states in simple surfactant aggregates

Excitation of a molecule from the ground state to an electronically excited state can cause changes in its geometry, dipole moment, acidity or basicity, redox potentials and solvation. Bimolecular quenching of the excited state of the probe by other molecules present in the medium can be used to determine the mobilities of molecules and estimate microviscosities and encounter probabilities in the medium. Differences in excited state acidity or basicity relative to the ground state can be employed to investigate the dynamics of ultrafast proton transfer reactions. Three areas of current interest where fluorescent probes have served to elucidate important dynamic processes of molecules in simple self-aggregating surfactant systems such as aqueous micelles and reverse micelles are considered: (a) bimolecular quenching of excited states; (b) the dynamics of solvation of excited states and (c) ultrafast intermolecular excited state proton transfer (ESPT) reactions.     

An alternate proton acceptor for excited-state proton transfer in green fluorescent protein: rewiring GFP

The neutral form of the chromophore in wild-type green fluorescent protein (wtGFP) undergoes excited-state proton transfer (ESPT) upon excitation, resulting in characteristic green (508 nm) fluorescence. This ESPT reaction involves a proton relay from the phenol hydroxyl of the chromophore to the ionized side chain of E222, and results in formation of the anionic chromophore in a protein environment optimized for the neutral species (the I* state). Reorientation or replacement of E222, as occurs in the S65T and E222Q GFP mutants, disables the ESPT reaction and results in loss of green emission following excitation of the neutral chromophore. Previously, it has been shown that the introduction of a second mutation (H148D) into S65T GFP allows the recovery of green emission, implying that ESPT is again possible. A similar recovery of green fluorescence is also observed for the E222Q/H148D mutant, suggesting that D148 is the proton acceptor for the ESPT reaction in both double mutants. The mechanism of fluorescence emission following excitation of the neutral chromophore in S65T/H148D and E222Q/H148D has been explored through the use of steady state and ultrafast time-resolved fluorescence and vibrational spectroscopy. The data are contrasted with those of the single mutant S65T GFP. Time-resolved fluorescence studies indicate very rapid (< 1 ps) formation of I* in the double mutants, followed by vibrational cooling on the picosecond time scale. The time-resolved IR difference spectra are markedly different to those of wtGFP or its anionic mutants. In particular, no spectral signatures are apparent in the picosecond IR difference spectra that would correspond to alteration in the ionization state of D148, leading to the proposal that a low-barrier hydrogen bond (LBHB) is present between the phenol hydroxyl of the chromophore and the side chain of D148, with different potential energy surfaces for the ground and excited states. This model is consistent with recent high-resolution structural data in which the distance between the donor and acceptor oxygen atoms is < or = 2.4 A. Importantly, these studies indicate that the hydrogen-bond network in wtGFP can be replaced by a single residue, an observation which, when fully explored, will add to our understanding of the various requirements for proton-transfer reactions within proteins.     

A DFT/TDDFT Study on Excited State Process of a Novel Probe 4′-Fluoroflavonol

A novel fluorescent probe 4′-fluoroflavonol (4F) was reported by Serdiuk et al. (RSC Adv 6:42532, 2016) in a previous paper. Spectroscopic studies on excited-state proton transfer (ESPT) of 4F was mentioned, while the mechanism of ESPT for 4F isdeficiency. In this present work, based on the time dependent density functional theory (TDDFT), we investigated the excited-state intramolecular proton transfer (ESIPT) mechanism of 4F theoretically. The primary bond lengths, bond angles and the infrared (IR) vibrational spectra involved in the formation of hydrogen bonds vertified the intramolecular hydrogen bond was strengthened, which manifests the tendency of excited state proton transfer. According to the results of calculated potential energy curves along O–H coordinate, an about 13.18 kcal/mol barrier has been found in the S₀ state. However, a barrier of 3.29 kcal/mol was found in the S₁ state, which demonstrates that the proton transfer process is more likely to occur in the excited state. In other words, the proton transfer was facilitated by photoexcitation. Particularly, the study about ESIPT mechanism of 4F should be helpful for further understanding property of fisetin.     

A theoretical investigation about the excited state behavior for 2‐(6'‐hydroxy‐2'‐pyridyl)benzimidazole: The water‐assisted excited state proton transfer process

In this work, based on density functional theory (DFT) and time‐dependent DFT (TD‐DFT) methods, we theoretically investigate the excited‐state process of the 2‐(6'‐hydroxy‐2'‐pyridyl)benzimidazole (2HPB) system in acetonitrile and water solvents. Since acetonitrile is an aprotic solvent, it has no effect on the solvent‐assisted excited‐state proton transfer (ESPT) process. Therefore, the 2HPB molecule cannot transfer the proton in acetonitrile, which is consistent with previous experimental observation. On the other hand, 2HPB can combine one water molecule (which is a protic solvent), forming the 2HPB–H₂O complex in the S₀ state. After photoexcitation, the intermolecular hydrogen bonds O1 H2···O3 and O3 H4···N5 both get strengthened in the S₁ state, which leads to the possibility of a water‐assisted ESPT process. Further, the charge redistribution reveals the tendency of ESPT. By exploring the potential energy curves for the 2HPB–H₂O complex in water, we confirm that a stepwise double proton transfer process occurs in the S₁ state. Water‐assisted ESIPT can occur along O1 H2···O3 or O3 H4···N5 because of their similar potential barriers. Based on the stepwise ESPT mechanism, we reinterpret the absorption and fluorescence spectra mentioned in the experiments and confirm the rationality of the water‐assisted ESPT process.     

Mechanistic aspects of proton chain transfer in the green fluorescent protein. Part II. A comparison of minimal quantum chemical models

In this paper we report the results of extensive quantum chemical reaction pathway calculations for the electronic ground state of several different cluster models that mimic the proton chain transfer path within the green fluorescent protein (GFP). Our principal objective is to establish the robustness with respect to variations in the model of our recent mechanistic inferences for the ground state proton chain transfer [S. Wang and S. C. Smith, J. Phys. Chem. B, 2006, 110, 5084]. Additionally, comparison of our ground state results with the excited state proton transfer (ESPT) study by Vendrell et al. [O. Vendrell, R. Gelabert, M. Moreno and J. M. Lluch, J. Am. Chem. Soc., 2006, 128, 3564] leads to the conclusion that the mechanism of proton chain transfer may be expected to be analogous in ground and excited states, principally because in both cases the loss of the chromophore's phenolic proton contributes strongly to the reaction coordinate only late in the reaction path.     

4-Azidocinnoline—Cinnoline-4-amine Pair as a New Fluorogenic and Fluorochromic Environment-Sensitive Probe

A new type of fluorogenic and fluorochromic probe based on the reduction of weakly fluorescent 4-azido-6-(4-cyanophenyl)cinnoline to the corresponding fluorescent cinnoline-4-amine was developed. We found that the fluorescence of 6-(4-cyanophenyl)cinnoline-4-amine is strongly affected by the nature of the solvent. The fluorogenic effect for the amine was detected in polar solvents with the strongest fluorescence increase in water. The environment-sensitive fluorogenic properties of cinnoline-4-amine in water were explained as a combination of two types of fluorescence mechanisms: aggregation-induced emission (AIE) and excited state intermolecular proton transfer (ESPT). The suitability of an azide–amine pair as a fluorogenic probe was tested using a HepG2 hepatic cancer cell line with detection by fluorescent microscopy, flow cytometry, and HPLC analysis of cells lysates. The results obtained confirm the possibility of the transformation of the azide to amine in cells and the potential applicability of the discovered fluorogenic and fluorochromic probe for different analytical and biological applications in aqueous medium.     

Excited-state proton transfer in N-methyl-6-hydroxyquinolinium salts: solvent and temperature effects

The excited-state proton transfer (ESPT) reaction of the "super"photoacid N-methyl-6-hydroxyquinolinium (MHQ) was studied using both fluorescence upconversion and time-correlated single photon counting (TCSPC) techniques. The ultrafast ESPT kinetics were investigated in various alcohols and water and determined to be solvent-controlled. The ESPT temperature dependence of MHQ was also studied in various alcohols and compared to that observed for another "super"photoacid, 5,8-dicyano-2-naphthol (DCN2). A full set of kinetic and thermodynamic parameters describing the ESPT was obtained. The protolytic photodissociation rate constant for MHQ was higher than that for DCN2, while the ESPT activation energies of MHQ were smaller. These findings are attributed to the approximately 3 orders of magnitude differences in excited-state acidities of MHQ and DCN2.     

Synthesis and spectroscopic studies of model red fluorescent protein chromophores

[reaction: see text]. Here we describe the synthesis and spectroscopic characterization of two compounds designed to model the chromophore in DsRed, a red fluorescent protein. Comparison with model green fluorescent protein (GFP) chromophores indicates that the additional conjugation in the DsRed models can account, in part, for the red-shifted absorption and emission properties of DsRed compared to those of GFP. In contrast to the GFP models, the DsRed models are fluorescent with quantum yields of 0.002-0.01 in CHCl3.     

Excited-state proton transfer via hydrogen-bonded acetic acid (AcOH) wire for 6-hydroxyquinoline

Spectroscopic studies on excited-state proton transfer (ESPT) of hydroxyquinoline (6HQ) have been performed in a previous paper. And a hydrogen-bonded network formed between 6HQ and acetic acid (AcOH) in nonpolar solvents has been characterized. In this work, a time-dependent density functional theory (TDDFT) method at the def-TZVP/B3LYP level was employed to investigate the excited-state proton transfer via hydrogen-bonded AcOH wire for 6HQ. A hydrogen-bonded wire containing three AcOH molecules at least for connecting the phenolic and quinolinic -N- group in 6HQ has been confirmed. The excited-state proton transfer via a hydrogen-bonded wire could result in a keto tautomer of 6HQ and lead to a large Stokes shift in the emission spectra. According to the results of calculated potential energy (PE) curves along different coordinates, a stepwise excited-state proton transfer has been proposed with two steps: first, an anionic hydrogen-bonded wire is generated by the protonation of -N- group in 6HQ upon excitation to the S(1) state, which increases the proton-capture ability of the AcOH wire; then, the proton of the phenolic group transfers via the anionic hydrogen-bonded wire, by an overall "concerted" process. Additionally, the formation of the anionic hydrogen-bonded wire as a preliminary step has been confirmed by the hydrogen-bonded parameters analysis of the ESPT process of 6HQ in several protic solvents. Therefore, the formation of anionic hydrogen-bonded wire due to the protonation of the -N- group is essential to strengthen the hydrogen bonding acceptance ability and capture the phenolic proton in the 6HQ chromophore.     

Excited-state proton transfer from pyranine to acetate in gamma-cyclodextrin and hydroxypropyl gamma-cyclodextrin

Excited-state proton transfer (ESPT) from pyranine (8-hydroxypyrene-1,3,6-trisulfonate, HPTS) to acetate has been studied by picosecond and femtosecond emission spectroscopy in gamma-cyclodextrin (gamma-CD) and 2-hydroxypropyl-gamma-cyclodextrin (HP-gamma-CD) cavities. In both the CDs, ESPT from HPTS to acetate is found to be very much slower (90 and 200 ps) than that in bulk water (0.15 and 6 ps). From molecular modeling, it is shown that in the cyclodextrin cavity the acetate is separated from the OH group of HPTS by water bridges. As a result, proton transfer in the cavity requires rearrangement of the hydrogen-bond network involving the cyclodextrin. This is responsible for the marked slowdown of ESPT. ESPT of HPTS in substituted gamma-CD is found to be slower than that in the unsubstituted one. This is attributed to the hydroxypropyl groups, which prevent close approach of acetate to HPTS.     

TD‐DFT study on the sensing mechanism of a fluorescent chemosensor for fluoride: Excited‐state proton transfer

An excited‐state proton transfer (ESPT) process, induced by both intermolecular and intramolecular hydrogen‐bonding interactions, is proposed to account for the fluorescence sensing mechanism of a fluoride chemosensor, phenyl‐1H‐anthra(1,2‐d)imidazole‐6,11‐dione. The time‐dependent density functional theory (TD‐DFT) method has been applied to investigate the different electronic states. The present theoretical study of this chemosensor, as well as its anion and fluoride complex, has been conducted with a view to monitoring its structural and photophysical properties. The proton of the chemosensor can shift to fluoride in the ground state but transfers from the proton donor (NH group) to a proton acceptor (neighboring carbonyl group) in the first singlet excited state. This may explain the observed red shifts in the fluorescence spectra in the relevant fluorescent sensing mechanism. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2010