Theoretical studies on the isomerization mechanism of the ortho-green fluorescent protein chromophore

We present a systematic theoretical investigation on the overall ground state and excited-state isomerization reaction mechanism of ortho-green fluorescent protein chromophore (o-HBDI) using the density functional theory and the multireference methods. The calculated results and subsequent analysis suggest the possible isomerization mechanism for o-HBDI. By comparison with experimental observation and detailed analysis, it is concluded that as initiated by the excited-state intramolecular proton transfer reaction, the conical intersection between the ground state and the excited state along the C4-C5 single-bond rotational coordinate is responsible for the rapid deactivation of o-HBDI.     

Deactivation mechanism of the green fluorescent chromophore

We report time-resolved fluorescence data for the anion of p-hydroxybenzylidene dimethylimidazolinone (p-HBDI), a model chromophore of the green fluorescence protein, in viscous glycerol-water mixtures over a range of temperatures, T. The markedly nonexponential decay of the excited electronic state is interpreted with the aid of an inhomogeneous model possessing a Gaussian coordinate-dependent sink term. A nonlinear least-squares fitting routine enables us to achieve quantitative fits by adjusting a single activation parameter, which is found to depend linearly on 1/T. We derive an analytic expression for the absolute quantum yield, which is compared with the integrated steady-state fluorescence spectra. The microscopic origins of the model are discussed in terms of two-dimensional dynamics, coupling the phenyl-ring rotation to a swinging mode that brings this flexible molecule to the proximity of a conical intersection on its multidimensional potential energy surface.     

Low-temperature chromophore isomerization reveals the photoswitching mechanism of the fluorescent protein Padron

Photoactivatable fluorescent proteins are essential players in nanoscopy approaches based on the super-localization of single molecules. The subclass of reversibly photoswitchable fluorescent proteins typically activate through isomerization of the chromophore coupled with a change in its protonation state. However, the interplay between these two events, the details of photoswitching pathways, and the role of protein dynamics remain incompletely understood. Here, by using a combination of structural and spectroscopic approaches, we discovered two fluorescent intermediate states along the on-switching pathway of the fluorescent protein Padron. The first intermediate can be populated at temperatures as low as 100 K and results from a remarkable trans-cis isomerization of the anionic chromophore taking place within a protein matrix essentially deprived of conformational flexibility. This intermediate evolves in the dark at cryotemperatures to a second structurally similar but spectroscopically distinct anionic intermediate. The final fluorescent state, which consists of a mixture of anionic and neutral chromophores in the cis configuration, is only reached above the glass transition temperature, suggesting that chromophore protonation involves solvent interactions mediated by pronounced dynamical breathing of the protein scaffold. The possibility of efficiently and reversibly photoactivating Padron at cryotemperatures will facilitate the development of advanced super-resolution imaging modalities such as cryonanoscopy.     

Excited-state structure determination of the green fluorescent protein chromophore

Ultrafast polarization-sensitive infrared (IR) spectroscopy of the C=O stretching mode of the chromophore of the green fluorescent protein reveals a near complete twisting around the ethylenic bridge between the phenolate and imidazolidinone groups upon electronic excitation, hinting at a decisive role of this motion in the efficient internal conversion process.     

Reaction progress of chromophore biogenesis in green fluorescent protein

The mature form of green fluorescent protein (GFP) is generated by a spontaneous self-modification process that is essentially irreversible. A key step in chromophore biosynthesis involves slow air oxidation of an intermediate species, in which the backbone atoms of residues 65-67 have condensed to form a five-membered heterocycle. We have investigated the kinetics of hydrogen peroxide evolution during in vitro GFP maturation and found that the H2O2 coproduct is generated prior to the acquisition of green fluorescence at a stoichiometry of 1:1 (peroxide/chromophore). The experimental progress curves were computer-fitted to a three-step mechanism, in which the first step proceeds with a time constant of 1.5 (+/-1.1) min and includes protein folding and peptide cyclization. Kinetic data obtained by HPLC analysis support a rapid cyclization reaction that can be reversed upon acid denaturation. The second step proceeds with a time constant of 34.0 (+/-1.5) min and entails rate-limiting protein oxidation, as supported by a mass loss of 2 Da observed for tryptic peptides derived from species that accumulate during the reaction. The final step in GFP maturation proceeds with a time constant of 10.6 (+/-1.2) min, suggesting that this step may contribute to overall rate retardation. We propose that under highly aerobic conditions, the dominant reaction path follows a cyclization-oxidation-dehydration mechanism, in which dehydration of the heterocycle is facilitated by slow proton abstraction from the Tyr66 beta-carbon. In combination, the results presented here suggest a role for molecular oxygen in trapping the cyclized form of GFP.     

Bond selection in the photoisomerization reaction of anionic green fluorescent protein and kindling fluorescent protein chromophore models

The chromophores of the most widely known fluorescent proteins (FPs) are derivatives of a core p-hydroxybenzylidene-imidazolinon-5-one (HBI) motif, which usually occurs as a phenolate anion. Double bond photoisomerization of the exocyclic bridge of HBI is widely held to be an important internal conversion mechanism for FP chromophores. Herein we describe the ground and excited-state electronic structures and potential energy surfaces of two model chromophores: 4- p-hydroxybenzylidiene-1,2-dimethyl-imidazolin-5-one anion (HBDI), representing green FPs (GFPs), and 2-acetyl-4-hydroxybenylidene-1-methyl-imidazolin-5-one anion (AHBMI), representing kindling FPs (KFPs). These chromophores differ by a single substitution, but we observe qualitative differences in the potential energy surfaces which indicate inversion of bond selection in the photoisomerization reaction. Bond selection is also modulated by whether the reaction proceeds from a Z or an E conformation. These configurations correspond to fluorescent and nonfluorescent states of structurally characterized FPs, including some which can be reversibly switched by specific illumination regimes. We explain the difference in bond selectivity via substituent stabilization effects on a common set of charge-localized chemical structures. Different combinations of these structures give rise to both optically active (planar) and twisted intramolecular charge-transfer (TICT) states of the molecules. We offer a prediction of the gas-phase absorption of AHBMI, which has not yet been measured. We offer a hypothesis to explain the unusual fluorescence of AHBMI in DMF solution, as well as an experimental proposal to test our hypothesis.     

Slow exchange in the chromophore of a green fluorescent protein variant

Green fluorescent protein and its mutants have become valuable tools in molecular biology. They also provide systems rich in photophysical and photochemical phenomena of which an understanding is important for the development of new and optimized variants of GFP. Surprisingly, not a single NMR study has been reported on GFPs until now, possibly because of their high tendency to aggregate. Here, we report the (19)F nuclear magnetic resonance (NMR) studies on mutants of the green fluorescent protein (GFP) and cyan fluorescent protein (CFP) labeled with fluorinated tryptophans that enabled the detection of slow molecular motions in these proteins. The concerted use of dynamic NMR and (19)F relaxation measurements, supported by temperature, concentration- and folding-dependent experiments provides direct evidence for the existence of a slow exchange process between two different conformational states of CFP. (19)F NMR relaxation and line shape analysis indicate that the time scale of exchange between these states is in the range of 1.2-1.4 ms. Thermodynamic analysis revealed a difference in enthalpy (Delta)H(0) = (18.2 +/- 3.8) kJ/mol and entropy T(Delta)S(0) = (19.6 +/- 1.2) kJ/mol at T = 303 K for the two states involved in the exchange process, indicating an entropy-enthalpy compensation. The free energy of activation was estimated to be approximately 60 kJ/mol. Exchange between two conformations, either of the chromophore itself or more likely of the closely related histidine 148, is suggested to be the structural process underlying the conformational mobility of GFPs. The possibility to generate a series of single-atom exchanges ("atomic mutations") like H --> F in this study offers a useful approach for characterizing and quantifying dynamic processes in proteins by NMR.     

Isomerization in fluorescent protein chromophores involves addition/elimination

The green fluorescent protein (GFP) chromophore undergoes both photochemical and thermal isomerizations. Typically, the Z form is more stable and undergoes photochemical conversion to the E form followed by thermal reversion over a period of seconds or minutes. Although the mechanism of the thermal reversion has been the subject of some investigations, the surprisingly low activation energy for this process has not sparked any controversy. We now show that the chromophore is surprisingly stable in both E and Z forms and that the facile thermal reversion is the result of a novel nucleophilic addition/elimination mechanism. This observation may have implications for the intervention of such processes, as well as blinking and kindling, in fluorescent proteins.     

Resolving the ultrafast dynamics of the anionic green fluorescent protein chromophore in water

The chromophore of the green fluorescent protein (GFP) is critical for probing environmental influences on fluorescent protein behavior. Using the aqueous system as a bridge between the unconfined vacuum system and a constricting protein scaffold, we investigate the steric and electronic effects of the environment on the photodynamical behavior of the chromophore. Specifically, we apply ab initio multiple spawning to simulate five picoseconds of nonadiabatic dynamics after photoexcitation, resolving the excited-state pathways responsible for internal conversion in the aqueous chromophore. We identify an ultrafast pathway that proceeds through a short-lived (sub-picosecond) imidazolinone-twisted (I-twisted) species and a slower (several picoseconds) channel that proceeds through a long-lived phenolate-twisted (P-twisted) intermediate. The molecule navigates the non-equilibrium energy landscape via an aborted hula-twist-like motion toward the one-bond-flip dominated conical intersection seams, as opposed to following the pure one-bond-flip paths proposed by the excited-state equilibrium picture. We interpret our simulations in the context of time-resolved fluorescence experiments, which use short- and long-time components to describe the fluorescence decay of the aqueous GFP chromophore. Our results suggest that the longer time component is caused by an energetically uphill approach to the P-twisted intersection seam rather than an excited-state barrier to reach the twisted intramolecular charge-transfer species. Irrespective of the location of the nonadiabatic population events, the twisted intersection seams are inefficient at facilitating isomerization in aqueous solution. The disordered and homogeneous nature of the aqueous solvent environment facilitates non-selective stabilization with respect to I- and P-twisted species, offering an important foundation for understanding the consequences of selective stabilization in heterogeneous and rigid protein environments.

Simulations on the aqueous green fluorescent protein (GFP) chromophore (in the equilibrium and non-equilibrium regimes) reveal that observed biexponential fluorescence originates from two competing torsional deactivation pathways.     

Photoactivation of the photoactive yellow protein: why photon absorption triggers a trans-to-cis Isomerization of the chromophore in the protein

Atomistic QM/MM simulations have been carried out on the complete photocycle of Photoactive Yellow Protein, a bacterial photoreceptor, in which blue light triggers isomerization of a covalently bound chromophore. The "chemical role" of the protein cavity in the control of the photoisomerization step has been elucidated. Isomerization is facilitated due to preferential electrostatic stabilization of the chromophore's excited state by the guanidium group of Arg52, located just above the negatively charged chromophore ring. In vacuo isomerization does not occur. Isomerization of the double bond is enhanced relative to isomerization of a single bond due to the steric interactions between the phenyl ring of the chromophore and the side chains of Arg52 and Phe62. In the isomerized configuration (ground-state cis), a proton transfer from Glu46 to the chromophore is far more probable than in the initial configuration (ground-state trans). It is this proton transfer that initiates the conformational changes within the protein, which are believed to lead to signaling.     

Comprehensive studies on an overall proton transfer cycle of the ortho-green fluorescent protein chromophore

Initiated by excited-state intramolecular proton transfer (ESIPT) reaction, an overall reaction cycle of 4-(2-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one (o-HBDI), an analogue of the core chromophore of the green fluorescent protein (GFP), has been investigated. In contrast to the native GFP core, 4-(4-hydroxybenzylidene)-1,2-dimethyl-1H-imidazol-5(4H)-one (p-HBDI), which requires hydrogen-bonding relay to accomplish proton transfer in vivo, o-HBDI possesses a seven-membered-ring intramolecular hydrogen bond and thus provides an ideal system for mimicking an intrinsic proton-transfer reaction. Upon excitation, ESIPT takes place in o-HBDI, resulting in a ～600 nm proton-transfer tautomer emission. The o-HBDI tautomer emission, resolved by fluorescence upconversion, is comprised of an instantaneous rise to a few hundred femtosecond oscillation in the early relaxation stage. Frequency analysis derived from ultrashort pulse gives two low-frequency vibrations at 115 and 236 cm(-1), corresponding to skeletal deformation motions associated with the hydrogen bond. The results further conclude that ESIPT in o-HBDI is essentially triggered by low-frequency motions and may be barrierless along the reaction coordinate. Femtosecond UV/vis transient absorption spectra also provide supplementary evidence for the structural evolution during the reaction. In CH(3)CN, an instant rise of a 530 nm transient is resolved, which then undergoes 7.8 ps decay, accompanied by the growth of a rather long-lived 580 nm transient species. It is thus concluded that following ESIPT the cis-proton transfer isomer undergoes cis-trans-isomerization. The results of viscosity-dependent dynamics are in favor of the one-bond-flip mechanism, which is in contrast to the volume-conserving isomerization behavior for cis-stilbene and p-HBDI. Further confirmation is given by the picosecond-femtosecond transient IR absorption spectra, where several new and long-lived IR bands in the range of 1400-1500 cm(-1) are assigned to the phenyl in-plane breathing motions of the trans-proton transfer tautomer. Monitored by the nanosecond transient absorption, the 580 nm transient undergoes a ～7.7 μs decay constant, accompanied by the growth of a new ～500 nm band. The latter is assigned to a deprotonated tautomer species, which then undergoes the ground-state reverse proton recombination to the original o-HBDI in ～50 μs, achieving an overall, reversible proton transfer cycle. This assignment is unambiguously supported by pump-probe laser induced fluorescence studies. On these standpoints, a comparison of photophysical properties among o-HBDI, p-HBDI, and wild-type GFP is discussed in detail.     

Absorption tuning of the green fluorescent protein chromophore: synthesis and studies of model compounds

Abstract  

The green fluorescent protein (GFP) chromophore is a heterocyclic compound containing a p-hydroxybenzylidine attached to an imidazol-5(4H)-one ring. This review covers the synthesis of a variety of model systems for elucidating the intrinsic optical properties of the chromophore in the gas phase and its response in particular to hydrogen bond interactions. The overall goal is to understand how the protein binding pocket influences the absorption behavior, and the current status of our ongoing efforts is presented.     

Radiationless decay of red fluorescent protein chromophore models via twisted intramolecular charge-transfer states

We use CASSCF and MRPT2 calculations to characterize the bridge photoisomerization pathways of a model red fluorescent protein (RFP) chromophore model. RFPs are homologues of the green fluorescent protein (GFP). The RFP chromophore differs from the GFP chromophore via the addition of an N-acylimine substitution to a common hydroxybenzylidene-imidazolinone (HBI) motif. We examine the substituent effects on the manifold of twisted intramolecular charge-transfer (TICT) states which mediates radiationless decay via bridge isomerization in fluorescent protein chromophore anions. We find that the substitution destabilizes states associated with isomerization about the imidazolinone-bridge bond and stabilizes states associated with phenoxy-bridge bond isomerization. We discuss the results in the context of chromophore conformation and quantum yield trends in the RFP subfamily, as well as recent studies on synthetic models where the acylimine has been replaced with an olefin.     

Excited-state intramolecular proton transfer molecules bearing o-hydroxy analogues of green fluorescent protein chromophore

o-Hydroxy analogues, 1a-g, of the green fluorescent protein chromophore have been synthesized. Their structures and electronic properties were investigated by X-ray single-crystal analyses, electrochemistry, and luminescence properties. In solid and nonpolar solvents 1a-g exist mainly as Z conformers that possess a seven-membered-ring hydrogen bond and undergo excited-state intramolecular proton transfer (ESIPT) reactions, resulting in a proton-transfer tautomer emission. Fluorescence upconversion dynamics have revealed a coherent type of ESIPT, followed by a fast vibrational/solvent relaxation (<1 ps) to a twisted (regarding exo-C(5)-C(4)-C(3) bonds) conformation, from which a fast population decay of a few to several tens of picoseconds was resolved in cyclohexane. Accordingly, the proton-transfer tautomer emission intensity is moderate (0.08 in 1e) to weak (～10(-4) in 1a) in cyclohexane. The stronger intramolecular hydrogen bonding in 1g suppresses the rotation of the aryl-alkene bond, resulting in a high yield of tautomer emission (Φ(f) ≈ 0.2). In the solid state, due to the inhibition of exo-C(5)-C(4)-C(3) rotation, intense tautomer emission with a quantum yield of 0.1-0.9 was obtained for 1a-g. Depending on the electronic donor or acceptor strength of the substituent in either the HOMO or LUMO site, a broad tuning range of the emission from 560 (1g) to 670 nm (1a) has been achieved.     

Design and concise synthesis of a novel type of green fluorescent protein chromophore analogue

A small molecular model compound for the green fluorescent protein chromophore was readily synthesized by a novel condensation reaction of (thio)imidate with imino-ester via an aziridine intermediate. This compound showed fluorescence in the solid and frozen solution states but not in the solution state. Its fluorescent property was successfully applied in the detection of dsDNA.     

Stalling chromophore synthesis of the fluorescent protein Venus reveals the molecular basis of the final oxidation step

Fluorescent proteins (FPs) have revolutionised the life sciences, but the mechanism of chromophore maturation is still not fully understood. Here we show that incorporation of a photo-responsive non-canonical amino acid within the chromophore stalls maturation of Venus, a yellow FP, at an intermediate stage; a crystal structure indicates the presence of O₂ located above a dehydrated enolate form of the imidazolone ring, close to the strictly conserved Gly67 that occupies a twisted conformation. His148 adopts an “open” conformation so forming a channel that allows O₂ access to the immature chromophore. Absorbance spectroscopy supported by QM/MM simulations suggests that the first oxidation step involves formation of a hydroperoxyl intermediate in conjunction with dehydrogenation of the methylene bridge. A fully conjugated mature chromophore is formed through release of H₂O₂, both in vitro and in vivo. The possibility of interrupting and photochemically restarting chromophore maturation and the mechanistic insights open up new approaches for engineering optically controlled fluorescent proteins.

Fluorescent proteins (FPs) have revolutionised the life sciences, but the chromophore maturation mechanism is still not fully understood. Here we photochemically trap maturation at a crucial stage and structurally characterise the intermediate.     

Facile synthesis of 4-arylidene-5-imidazolinones as synthetic analogs of fluorescent protein chromophore

A facile and effective synthesis for a wide variety of 4-arylidene-5-imidazolinone derivatives was developed. 4-Arylidene-5-oxazolinones were prepared by Erlenmeyer azlactone synthesis from N-acylglycines and arylaldehydes. The ring-opening reactions of the 4-arylidene-5-oxazolinones with primary amines afforded 2-acylamino-3-arylacrylamides in excellent yields. A new dehydrative cyclization of the 2-acylamino-3-arylacrylamides in pyridine under reflux furnished the corresponding 4-arylidene-5-imidazolinones in good yields.     

Origin, nature, and fate of the fluorescent state of the green fluorescent protein chromophore at the CASPT2//CASSCF resolution

Ab initio CASPT2//CASSCF relaxation path computations are employed to determine the intrinsic (e.g., in vacuo) mechanism underlying the rise and decay of the luminescence of the anionic form of the green fluorescent protein (GFP) fluorophore. Production and decay of the fluorescent state occur via a two-mode reaction coordinate. Relaxation along the first (totally symmetric) mode leads to production of the fluorescent state that corresponds to a planar species. The second (out-of-plane) mode controls the fluorescent state decay and mainly corresponds to a barrierless twisting of the fluorophore phenyl moiety. While a "space-saving" hula-twist conical intersection decay channel is found to lie only 5 kcal mol(-1) above the fluorescent state, the direct involvement of a hula-twist deformation in the decay is not supported by our data. The above results indicate that the ultrafast fluorescence decay observed for the GFP chromophore in solution is likely to have an intrinsic origin. The possible effects of the GFP protein cavity on the fluorescence lifetime of the investigated chromophore model are discussed.