Green Fluorescent Protein Chromophore Anion Research Articles

Chemical control of excited-state reactivity of the anionic green fluorescent protein chromophore.

Controlling excited-state reactivity is a long-standing challenge in photochemistry, as a desired pathway may be inaccessible or compete with other unwanted channels. An important example is internal conversion of the anionic green fluorescent protein (GFP) chromophore where non-selective progress along two competing torsional modes (P: phenolate and I: imidazolinone) impairs and enables Z-to-E photoisomerization, respectively. Developing strategies to promote photoisomerization could drive new areas of applications of GFP-like proteins. Motivated by the charge-transfer dichotomy of the torsional modes, we explore chemical substitution on the P-ring of the chromophore as a way to control excited-state pathways and improve photoisomerization. As demonstrated by methoxylation, selective P-twisting appears difficult to achieve because the electron-donating potential effects of the substituents are counteracted by inertial effects that directly retard the motion. Conversely, these effects act in concert to promote I-twisting when introducing electron-withdrawing groups. Specifically, 2,3,5-trifluorination leads to both pathway selectivity and a more direct approach to the I-twisted intersection which, in turn, doubles the photoisomerization quantum yield. Our results suggest P-ring engineering as an effective approach to boost photoisomerization of the anionic GFP chromophore.

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.

A photoelectron imaging study of the deprotonated GFP chromophore anion and RNA fluorescent tags.

Green fluorescent protein (GFP), together with its family of variants, is the most widely used fluorescent protein for in vivo imaging. Numerous spectroscopic studies of the isolated GFP chromophore have been aimed at understanding the electronic properties of GFP. Here, we build on earlier work [A. V. Bochenkova, C. Mooney, M. A. Parkes, J. Woodhouse, L. Zhang, R. Lewin, J. M. Ward, H. Hailes, L. H. Andersen and H. H. Fielding, Chem. Sci., 2017, 8, 3154] investigating the impact of fluorine and methoxy substituents that have been employed to tune the electronic structure of the GFP chromophore for use as fluorescent RNA tags. We present photoelectron spectra following photoexcitation over a broad range of wavelengths (364-230 nm) together with photoelectron angular distributions following photoexcitation at 364 nm, which are interpreted with the aid of quantum chemistry calculations. The results support the earlier high-level quantum chemistry calculations that predicted how fluorine and methoxy substituents tune the electronic structure and we find evidence to suggest that the methoxy substituents enhance internal conversion, most likely from the 2ππ* state which has predominantly Feshbach resonance character, to the 1ππ* state.

Ab-initio molecular dynamics and hybrid explicit-implicit solvation model for aqueous and nonaqueous solvents: GFP chromophore in water and methanol solution as case study.

Solute-solvent interactions are proxies for understanding how the electronic density of a chromophore interacts with the environment in a more exhaustive way. The subtle balance between polarization, electrostatic, and non-bonded interactions need to be accurately described to obtain good agreement between simulations and experiments. First principles approaches providing accurate configurational sampling through molecular dynamics may be a suitable choice to describe solvent effects on solute chemical-physical properties and spectroscopic features, such as optical absorption of dyes. In this context, accurate energy potentials, obtained by hybrid implicit/explicit solvation methods along with employing nonperiodic boundary conditions, are required to represent bulk solvent around a large solute-solvent cluster. In this work, a novel strategy to simulate methanol solutions is proposed combining ab initio molecular dynamics, a hybrid implicit/explicit flexible solvent model, nonperiodic boundary conditions, and time dependent density functional theory. As case study, the robustness of the proposed protocol has been gauged by investigating the microsolvation and electronic absorption of the anionic green fluorescent protein chromophore in methanol and aqueous solution. Satisfactory results are obtained, reproducing the microsolvation layout of the chromophore and, as a consequence, the experimental trends shown by the optical absorption in different solvents.

Quantum mechanical/molecular mechanical trajectory surface hopping molecular dynamics simulation by spin-flip time-dependent density functional theory.

This paper presents the nonadiabatic molecular dynamics simulation in the solution phase using the spin-flip time-dependent density functional theory (SF-TDDFT). Despite the single-reference level of theory, the SF-TDDFT method can generate the correct topology of S0/S1 crossing points, thus providing a natural S1 → S0 nonadiabatic transition. We extend the gas-phase trajectory surface hopping simulation with the SF-TDDFT [N. Minezawa and T. Nakajima, J. Chem. Phys. 150, 204120 (2019)] to the hybrid quantum mechanical/molecular mechanics (QM/MM) scheme. To this end, we modify the code to evaluate the electrostatic interaction between the QM and MM atoms and to extract the classical MM energy and forces from the MM program package. We apply the proposed method to the photoisomerization reaction of aqueous E-azomethane and anionic green fluorescent protein chromophore in water and compare the results with those of the previous simulation studies based on the multireference methods.

The Effect of an Electric Field on the Spectroscopic Properties of the Isolated Green Fluorescent Protein Chromophore Anion

AbstractThe front cover artwork is provided by the groups of Profs. Lars H. Andersen and Steen Brøndsted Nielsen, both at Aarhus University. The image shows the Green Fluorescent Protein as a jigsaw puzzle. The chromophore is colored blue and experiences a high electric field at one end. Read the full text of the Communication at 10.1002/cphc.201800225.

Front Cover: The Effect of an Electric Field on the Spectroscopic Properties of the Isolated Green Fluorescent Protein Chromophore Anion (ChemPhysChem 14/2018)

Front Cover: The Effect of an Electric Field on the Spectroscopic Properties of the Isolated Green Fluorescent Protein Chromophore Anion (ChemPhysChem 14/2018)

The Effect of an Electric Field on the Spectroscopic Properties of the Isolated Green Fluorescent Protein Chromophore Anion.

Here we uncover the direct effect of a high electric field on the absorption by the Green Fluorescent Protein chromophore anion isolated in vacuo based on gas-phase action spectroscopy. Betaine is a strong molecular dipole that creates an electric field of ∼70 MV/cm when attached to the ion at the phenolate oxygen, more than half the actual field from the protein matrix and pointing in the same direction. Nevertheless, the shift in absorption is limited (0.08 eV), supporting earlier conclusions, but subject to much debate, that the protein is rather innocent in perturbing the transition energy. The betaine complexes are readily made by electrospray ionization and in contrast to the bare ions, they dissociate after one-photon absorption. Also, electron detachment is not an open channel complicating the bare ion case. As steric constraints are absent in vacuo, the possibility of turning on fluorescence by an electric field can be tested from experiments on complexes with betaine.

Direct monitoring of photon induced isomerization, dissociation and electron detachment of the green fluorescent protein chromophore anion

SynopsisWe present the first experimental demonstration of Z↔E photoisomerization the GFP chromophore anion, HBDI−, in the gas phase. In the single photon absorption regime, the photoisomerization action spectra show two maxima at 480 nm and 455 nm. In the multiphoton absorption regime, photodissociation and photodetachment channels modify the appearance of the photoisomerization band. This work provides a new approach to characterize photoisomerization pathways in biomolecular ions.

Ortho and para chromophores of green fluorescent protein: controlling electron emission and internal conversion.

Green fluorescent protein (GFP) continues to play an important role in the biological and biochemical sciences as an efficient fluorescent probe and is also known to undergo light-induced redox transformations. Here, we employ photoelectron spectroscopy and quantum chemistry calculations to investigate how the phenoxide moiety controls the competition between electron emission and internal conversion in the isolated GFP chromophore anion, following photoexcitation with ultraviolet light in the range 400-230 nm. We find that moving the phenoxide group from the para position to the ortho position enhances internal conversion back to the ground electronic state but that adding an additional OH group to the para chromophore, at the ortho position, impedes internal conversion. Guided by quantum chemistry calculations, we interpret these observations in terms of torsions around the C-C-C bridge being enhanced by electrostatic repulsions or impeded by the formation of a hydrogen-bonded seven-membered ring. We also find that moving the phenoxide group from the para position to the ortho position reduces the energy required for detachment processes, whereas adding an additional OH group to the para chromophore at the ortho position increases the energy required for detachment processes. These results have potential applications in tuning light-induced redox processes of this biologically and technologically important fluorescent protein.

Probing microhydration effect on the electronic structure of the GFP chromophore anion: Photoelectron spectroscopy and theoretical investigations.

The photophysics of the Green Fluorescent Protein (GFP) chromophore is critically dependent on its local structure and on its environment. Despite extensive experimental and computational studies, there remain many open questions regarding the key fundamental variables that govern this process. One outstanding problem is the role of autoionization as a possible relaxation pathway of the excited state under different environmental conditions. This issue is considered in our work through combined experimental and theoretical studies of microsolvated clusters of the deprotonated p-hydroxybenzylidene-2,3-dimethylimidazolinone anion (HBDI(-)), an analog of the GFP chromophore. Through selective generation of microsolvated structures of predetermined size and subsequent analysis of experimental photoelectron spectra by high level ab initio methods, we are able to precisely identify the structure of the system, establish the accuracy of theoretical data, and provide reliable description of auto-ionization process as a function of hydrogen-bonding environment. Our study clearly illustrates the first few water molecules progressively stabilize the excited state of the chromophore anion against the autodetached neutral state, which should be an important trait for crystallographic water molecules in GFPs that has not been fully explored to date.

Excited state dynamics of the isolated green fluorescent protein chromophore anion following UV excitation.

A combined frequency-, angle-, and time-resolved photoelectron spectroscopy study is used to unravel the excited state dynamics following UV excitation of the isolated anionic chromophore of the green fluorescent protein (GFP). The optically bright S3 state, which is populated for hv > 3.7 eV, is shown to decay predominantly by internal conversion to the S2 state that in turn autodetaches to the neutral ground state. For hv > 4.1 eV, a new and favorable autodetachment channel from the S2 state becomes available, which leads to the formation of the neutral in an excited state. The results indicate that the UV excited state dynamics of the GFP chromophore involve a number of strongly coupled excited states.

Non-adiabatic dynamics of isolated green fluorescent protein chromophore anion.

On-the-fly ab initio molecular dynamics calculations have been performed to investigate the relaxation mechanism of green fluorescent protein chromophore anion under vacuum. The CASSCF surface hopping simulation method based on Zhu-Nakamura theory is applied to present the real-time conformational changes of the target molecule. The static calculations and dynamics simulation results suggest that not only the twisting motion around bridging bonds between imidazolinone and phenoxy groups but the strength mode of C=O and pyramidalization character of bridging atom are major factors on the ultrafast fluorescence quenching process of the isolated chromophore anion. The abovementioned factors bring the molecule to the vicinity of conical intersections on its potential energy surface and to finish the internal conversion process. A Hula-like twisting pattern is displayed during the relaxation process and the entire decay process disfavors a photoswitching pattern which corresponds to cis-trans photoisomerization.

Diabatic Population Matrix Formalism for Performing Molecular Mechanics Style Simulations with Multiple Electronic States.

An accurate description of nonbonded interactions is important in investigating dynamics of molecular systems. In many situations, fixed point charge models are successfully applied to explaining various chemical phenomena. However, these models with conventional formulations will not be appropriate in elucidating the detailed dynamics during nonadiabatic events. This is mainly because the chemical properties of any molecule, especially its electronic populations, significantly change with respect to molecular distortions in the vicinity of the surface crossing. To overcome this issue in molecular simulations yet within the framework of the fixed point charge model, we define a diabatic electronic population matrix and substitute it for the conventional adiabatic partial charges. We show that this matrix can be readily utilized toward attaining more reliable descriptions of Coulombic interactions, in combination with the interpolation formalism for obtaining the intramolecular interaction potential. We demonstrate how the mixed formalism with the diabatic charges and the interpolation can be applied to molecular simulations by conducting adiabatic and nonadiabatic molecular dynamics trajectory calculations of the green fluorescent protein chromophore anion in aqueous environment.

Competition between photodetachment and autodetachment of the 2(1)ππ* state of the green fluorescent protein chromophore anion.

Using a combination of photoelectron spectroscopy measurements and quantum chemistry calculations, we have identified competing electron emission processes that contribute to the 350-315 nm photoelectron spectra of the deprotonated green fluorescent protein chromophore anion, p-hydroxybenzylidene-2,3-dimethylimidazolinone. As well as direct electron detachment from S0, we observe resonant excitation of the 2(1)ππ* state of the anion followed by autodetachment. The experimental photoelectron spectra are found to be significantly broader than photoelectron spectrum calculated using the Franck-Condon method and we attribute this to rapid (∼10 fs) vibrational decoherence, or intramolecular vibrational energy redistribution, within the neutral radical.

On the optical absorption of the anionic GFP chromophore in vacuum, solution, and protein

In spite of the large number of experimental and theoretical studies, the optical absorption trend of the green fluorescent protein (GFP) chromophore in several environments has not been fully understood. We calculated at the same level of time dependent density functional theory the vertical excitation energy of the anionic GFP chromophore in the protein and in ethanol, dioxane, methanol and water solutions. As result, we reproduced for the first time the experimental trend of the absorption peaks with 0.015 eV as the standard deviation of the accuracy. This systematic error allowed us to analyze with confidence the relative weight of several solvation effects on the vertical excitation energy. Experimental trends not correlated with the solvent polarity were therefore explained with a fine balance of different steric and electronic effects on the photophysics of the chromophore. As an indirect and remarkable result, the present analysis confirms that the optical absorption of the chromophore in the gas phase is close to the value of 2.84 eV extrapolated by Dong et al. (J. Am. Chem. Soc., 2006, 128, 12038), and, as a consequence, that the protein environment induces a red shift of 0.23 eV.

Taking the green fluorescence out of the protein: dynamics of the isolated GFP chromophore anion

The green fluorescent protein (GFP) is employed extensively as a marker in biology and the life sciences as a result of its spectacular fluorescence properties. Here, we employ femtosecond time-resolved photoelectron spectroscopy to investigate the ultrafast excited state dynamics of the isolated GFP chromophore anion. Excited state population is found to decay bi-exponentially, with characteristic lifetimes of 330 fs and 1.4 ps. Distinct photoelectron spectra can be assigned to each of these timescales and point to the presence of a transient intermediate along the decay coordinate. Guided by ab initio calculations, we assign these observations to twisting about the C–C–C bridge followed by internal conversion to the anion ground state. The dynamics in vacuo are very similar to those observed in solution, despite the difference in absorption spectra between the two media. This is consistent with the protein environment restricting rotation about the C–C–C bond in order to prevent ultrafast internal conversion and preserve the fluorescence.

Ultrafast dual photoresponse of isolated biological chromophores: link to the photoinduced mode-specific non-adiabatic dynamics in proteins

The anionic wild-type Green Fluorescent Protein (GFP) chromophore defines an entire class of naturally occurring chromophores, which are based on the oxydized tyrosine side chain. The GFP chromophore exhibits an enriched photoinduced non-adiabatic dynamics in the multiple excited-state decay channels. Deactivation includes vibrational resonant photodetachment and internal conversion. Here, we provide detailed insight into the efficiency of different vibrational modes in promoting a selective photoresponse in the bare GFP chromophore anion. We introduce a general theoretical model that is capable of accounting for the alternative non-equivalent pathways in internal conversion, and we outline the factors, by which the photo-initiated response may be altered in this channel. The topography around the planar minimum in S1 and the two distinct types of the S1/S0 conical intersections obtained through high-level ab initio calculations provide direct support to the proposed model. There are mode-selective ways to control the photoresponse and to direct it towards a single excited-state decay channel. By tuning the excitation wavelength, the photoresponse may be directed towards the ultrafast non-statistical electron emission coupled with vibrational (de)coherence, whereas a vibrational pre-excitation in the ground state may lead to the ultrafast non-statistical internal conversion through a conical intersection. We also discuss the implication of our results to the photo-initiated non-adiabatic dynamics in the proteins.

Photodetachment Spectra of Deprotonated Fluorescent Protein Chromophore Anions

Isolated model anion chromophores of the green and cyan fluorescent proteins were generated in an electrospray ion source, and their photodetachment spectra were recorded using photoelectron imaging. Vertical photodetachment energies of 2.85(10) and 4.08(10) eV have been measured for the model green fluorescent protein chromophore anion, corresponding to photodetachment from the ground electronic state of the anion to the ground and first excited electronic states of the radical, respectively. For the model cyan fluorescent protein chromophore anion, vertical photodetachment energies of 2.88(10) and 3.96(10) eV have been measured, corresponding to detachment from the ground electronic state of the anion to the ground and first excited electronic states of the neutral radical, respectively. We also find evidence suggesting that autoionization of electronically excited states of the chromophore anions competes with direct photodetachment. For comparison and to benchmark our measurements, the vertical photodetachment energies of deprotonated phenol and indole anions have also been recorded and presented. Quantum chemistry calculations support our assignments. We discuss our results in the context of the isolated protein chromophore anions acting as electron donors, one of their potential biological functions.

Absorption and fluorescence spectra of the neutral and anionic green fluorescent protein chromophore: Franck–Condon simulation

Absorption and fluorescence spectra of the neutral and anionic green fluorescent protein (GFP) chromophore, namely p-hydroxybenzylideneimidazolidinone (p-HBDI), have been simulated using the Franck–Condon factors including inhomogeneous broadening of solvent effect. Ground and the first excited states were calculated by time dependent density functional theory with and without the polarizable continuum model environment. Simulated peak of the neutral/anionic p-HBDI at 380nm (423nm)/421nm agrees with experiment value 370nm (434nm)/419nm for absorption (fluorescence) spectrum. Simulated width of the neutral/anionic p-HBDI at 0.51eV (0.54eV)/0.57eV agrees with experiment value 0.54eV (0.66eV)/0.56eV for absorption (fluorescence) spectrum.