Fluorescent Protein Chromophore Research Articles

Temperature Controlled Decay and Pendulum Dynamics of Green Fluorescent Protein (GFP) Chromophore.

The excited-state dynamics of the GFP chromophore, HBDI- (anionic p-hydroxybenzylidene-2,3-dimethylimidazolinone), were investigated through a combination of theoretical nonadiabatic molecular dynamics (NAMD) simulations and femtosecond transient absorption spectroscopy (fs-TA). The NAMD simulations revealed that the primary dynamics in excited states involve the formation of a P-twisted intermediate (S1min,P), which undergoes pendulum-like oscillations with respect to ϕ = 90°. This motion serves as a reservoir for the excited-state population and the primary source of fluorescence. Rather than a direct channel from the major S1min,P, a coordinated pathway of S1min,P → S1min → S1min,I → S0 is responsible for the decay to the ground state, emphasizing the importance of planar intermediate (S1min) formation. The experimental fs-TA spectra confirmed these dynamics, revealing three distinct time scales (340-470 fs, 1.4 ps, and 8.3 ps), corresponding to the formation of S1min,P and its decay governed by the coordinated pathway. At low temperatures, the coordinated decay pathway is suppressed, leading to prolonged fluorescence lifetimes, consistent with low-temperature experimental results. This study presents a new model for the excited-state dynamics of GFP chromophore, suggesting that pendulum motion and the coordinated decay pathway play a crucial role in regulating fluorescence intensity.

Revealing the Role of Electronic Effect to Modulate the Photophysics and Z-Scan Responses of o-Locked GFP Chromophores.

Three novel core green fluorescent protein (GFP) chromophore analogues, based on a doubly locked conformation and variable electronic effects by replacing one hydrogen with bromine, iodine, and methyl, respectively, have been synthesized to modulate the push-pull effect. These chromophores exhibited intramolecular H-bonding, as evidenced by single-crystal X-ray and 1H NMR studies. The fluorescence quantum yields (ϕf) of all of the chromophores were found to be more than an order of magnitude higher (∼0.2) than the unlocked chromophores (∼0.01). It was found that the electronic effect did affect the nonradiative rates, as the quantum yields were found to vary with respect to different analogues in the same solvents. The effect of the push-pull effect was also evident by a higher Stokes-shifted emission in the case of the methyl derivative with respect to the bromo- and iodo-analogues. Furthermore, the emission spectra of these GFP chromophores were found to show positive solvatochromism, which was supported by a quantum chemical calculation. A detailed study, correlating the observed spectral changes with various solvent functions and supported by computational results, established a facile proton transfer, followed by twisted intramolecular charge transfer (TICT) to be the major nonradiative channels of these chromophores. Besides, a completely novel usage of these chromophores was explored for the first time by studying their third-order nonlinear optical characteristics in DMSO using a single-beam Z-scan technique. All of the chromophores exhibited tunable nonlinear refraction (NLR) and nonlinear absorption (NLA) properties that depend upon different substituent groups present in the chromophores. Here, the NLR was due to the effect of self-defocusing, whereas the NLA was triggered by reverse saturable absorption, which is attributed to the two-photon absorption (TPA) process. Surprisingly, the efficiency of the TPA ability of the chromophores was found to be a function of the induced electronic effect. Hence, this work opens a new route for the utility of the ortho-locked GFP chromophores in the field of nonlinear optical applications.

Determination of the Dipole Moment Variation Upon Excitation in the Chromophore of Green Fluorescent Protein From Molecular Dynamic Trajectories with QM/MM Potentials Using Machine Learning Methods

Quantum and molecular mechanics (QM/MM) potentials are used to calculate molecular dynamics trajectories for the EYFP protein of the green fluorescent protein family. Machine learning models are constructed to establish the relationship between the geometric parameters of the chromophore in the frame of its trajectory and the properties of its electronic excitation. It is shown that it is not enough to use only bridging bonds between the phenyl and imidazolidone fragments of the chromophore as a geometric parameter, and at least two more neighboring bonds must be added to the model. The proposed models allow determination of the dipole moment variation upon excitation with an average error of 0.11 a.u.

Self-assembly of green fluorescent protein chromophore analogue to form microfibers: Inhibited and disassembled by Hg2+ through Z-E isomerization

A novel analogue of the green fluorescent protein (GFP) chromophore, named p-DBCI, was synthesized via a 2,3-cycloaddition reaction. The compound was structurally characterized using NMR, HRMS, and single crystal X-ray diffraction, confirming its Z-form conformation in both solid and solution phases. While exhibiting low fluorescence quantum yields in solution, p-DBCI showed strong fluorescence emission in the solid state, indicating aggregation-induced emission (AIE) properties. p-DBCI demonstrated a remarkable ability for self-assembly, forming regular microrods with intense fluorescence through a solvent exchange method. The presence of Hg2+ was found to inhibit and disassemble the self-assembly of p-DBCI, leading to fluorescence quenching via Z-E isomerization driven by strong complexation between p-DBCI and Hg2+. The ability of Hg2+ to induce Z-E isomerization in GFP analogs has been rarely reported. Furthermore, the fluorescence intensity of p-DBCI assemblies in water solution exhibited a highly linear relationship (R2= 0.9992) with increasing concentrations of Hg2+, suggesting that p-DBCI could serve as a sensitive probe for quantitatively detecting Hg2+ in aqueous solutions. These findings offer new theoretical guidance for exploring and utilizing the solid fluorescence properties of GFP analogues.

GFP Chromophore Integrated Conjugated Microporous Polymers toward Bioinspired Photocatalytic CO2 Reduction to CO.

The development of highly active, durable, and low-cost metal-free catalysts for the photocatalytic CO2 reduction reaction (CO2RR) is an efficient and environmentally friendly solution to address significant problems like global warming and high energy demand. In the present study, we have demonstrated the design and synthesis of a donor-acceptor based conjugated microporous polymer (CMP), TPA-GFP, by integrating an electron donor, tris(4-ethynylphenyl)amine (TPA), with a green fluorescent protein chromophore analogue (Z)-4-(2-hydroxy-3,5-diiodobenzylidene)-1-(4-iodophenyl)-2-methyl-1H-imidazol-5(4H)-one (o-HBDI-I3) (GFP). In comparison to nondonor 1,3,5-triethynylbenzene (TEB) based TEB-GFP CMP, photocatalytic CO2 reduction using donor-acceptor based TPA-GFP CMP displays a 3-fold increment of CO production yield with a maximum CO yield of 1666 μmol g-1 at 12 h. Further, the CO selectivity increases significantly from a mere 54% in TEB-GFP to an impressive 95% in TPA-GFP. The impressive CO2 reduction efficiency and selectivity for TPA-GFP can be attributed to the efficient light-harvesting capability and facile charge separation and migration through donor-acceptor building units of the CMP. The mechanistic aspect of the photocatalytic CO2 reduction process is explored using in situ DRIFTS and DFT calculation, and a plausible photocatalytic mechanism is proposed.

Ultrafast Hot Exciton Nonadiabatic Excited-State Dynamics in Green Fluorescent Protein Chromophore Analogue.

The ultrafast high-energy nonadiabatic excited-state dynamics of the benzylidenedimethylimidazolinone chromophore dimer has been investigated using an electronic structure method coupled with on-the-fly quantitative wave function analysis to gain insight into the photophysics of hot excitons in biological systems. The dynamical simulation provides a rationalization of the behavior of the exciton in a dimer after the photoabsorption of light to higher-energy states. The results suggest that hot exciton localization within the manifold of excited states is caused by the hindrance of torsional rotation due to imidazolinone (I) or phenolate (P) bonds i.e., ΦI- or ΦP-dihedral rotation, in the monomeric units of a dimer. This hindrance arises due to weak π-π stacking interaction in the dimer, resulting in an energetically uphill excited-state barrier for ΦI- and ΦP-twisted rotation, impeding the isomerization process in the chromophore. Thus, this study highlights the potential impact of the weak π-π interaction in regulating the photodynamics of the green fluorescent protein chromophore derivatives.

Enzyme-responsive aptasensor based on the functionalized fluorescent protein chromophore derivative for the detection of alkaline phosphatase activity

Fluorescent proteins (FPs) based sensing strategies have been widely developed in bioanalytical applications. However, it still remains a challenge to realize the efficient and precise analysis. Recently, FPs chromophore and its derivatives with favorable optical properties are well-designed to simulate and replace the function of FPs by specifically binding to the selected fluorescent light-up aptamers (FLAPs), it is worthy note that there is still a lack of systematic approaches to remold these FPs mimics with enzyme-responsive functions. Taking alkaline phosphatase (ALP) as a research model, we here propose an ALP-responsive fluorescent DNA aptasensor (p-DAA) by utilizing a truncated Lettuce (tLet-10) FLAP and a phosphorylated DFHBI-derivative (ALP-DFSS) for achieving ALP activity analysis. Taking the HBI-chromophore as the remolding molecular template, ALP-DFSS has been successfully synthesized and characterized to response to ALP activity. With phosphate group substitution in ALP-DFSS, the decayed fluorescence of p-DAA can be observed by forming the complex of tLet-10/ALP-DFSS. While the turn-on fluorescence of p-DAA will be obtained by the ALP-triggered dephosphorylation process and further exhibit the label-free, fast-response, specific, and sensitive detection performances for both evaluating ALP activity and the inhibition efficiency of ALP inhibitor. Moreover, p-DAA allow the spiked ALP analysis in serum samples and is also applied for realizing the visual differentiation between normal cell line and cancer cell line by responding the endogenous ALP, which holds the great potential in guiding the chromophores functionalization and exploring aptasensing related biological research.

Viscosity-Induced Emission of 5-(Diarylmethylene)imidazolone with Extended Conjugation via Attachment of N-Methylpyrrole at the 2-Position.

We have developed a series of 2-monoaryl-5-diarylmethylene analogs of the green fluorescent protein chromophore to study their viscosity-induced emission (VIE) properties. The analogs were synthesized by a condensation with methyl imidate and N-(diarylmethylene)glycinate. Among the analogs, the N-methylpyrrol-2-yl-substituted analog 1h induced the most remarkable VIE behavior in triglyceride and lipid bilayers probably due to the high π-electron-rich property of the pyrrole ring. The pyrrole substituent in imidazolone analogs can be expected to become a common template for introducing VIE behavior.

Positively charged GFP-chromophore-based fluorescent sensor for DNA

Fluorescence titration of the positively charged green fluorescent protein (GFP) chromophore analogue 1 with E. Coli plasmid DNA [pcDNA3.1(−)] confirms that 1 serves as a fluorescent probe for sensing E. Coli plasmid DNA. The binding constant K between the DNA and 1 is as large as 2.9 × 105. The sensing mechanism is that the intermolecular steric hindrance generated between 1 and E. Coli plasmid DNA by electrostatic attraction restricts the φ-torsion around P-bond and τ-torsion around I-bond of 1, enhancing fluorescence of 1.

Substituent Effects in the Cationic Green Fluorescent Protein Chromophore: Ultrafast Excited‐State Proton Transfer or Twisting?

AbstractUnderstanding the structure‐function relationships of the green fluorescent protein (GFP) chromophore is important in rationally developing new molecular tools for biological imaging and beyond. Herein, we systematically modified the GFP chromophore structure with electron‐withdrawing and ‐donating groups (EWGs and EDGs) to investigate the substituent effects on the excited‐state proton transfer (ESPT) and twisting dynamics of the cationic chromophore in solution. With key insights gained from femtosecond transient absorption and stimulated Raman spectroscopy, we reveal that the EWG substitution by –F increases photoacidity in an additive manner and leads to an ultrafast barrierless ESPT by difluorination, while the EDG substitution by –OCH3 also results in ultrafast ESPT despite the weak photoacidity as estimated by the Förster equation. We ascribe the unusually fast kinetics in methoxylated derivatives to the occurrence of a pre‐existing chromophore‐solvent complex that sets up the acceptor site for ESPT. Furthermore, the kinetic competition between ESPT and twisting pathways is crucial for the observation of ESPT in action, particularly for molecules undergoing efficient nonradiative decay in the excited state through torsional motions. Such flexible and highly engineerable molecules can enable more versatile photoswitches and sensors.

Nuclear Quantum Effects on Nonadiabatic Dynamics of a Green Fluorescent Protein Chromophore Analogue: Ring-Polymer Surface-Hopping Simulation.

Herein, we have used the "on-the-fly" ring-polymer surface-hopping simulation method with the centroid approximation (RPSH-CA), in combination with the multireference OM2/MRCI electronic structure calculations to study the photoinduced dynamics of a green fluorescent protein (GFP) chromophore analogue in the gas phase, i.e., o-HBI, at 50, 100, and 300 K with 1, 5, 10, and 15 beads (3600 1 ps trajectories). The electronic structure calculations identified five new minimum-energy conical intersection (MECI) structures, which, together with the previous one, play crucial roles in the excited-state decay dynamics of o-HBI. It is also found that the excited-state intramolecular proton transfer (ESIPT) occurs in an ultrafast manner and is completed within 20 fs in all the simulation conditions because there is no barrier associated with this ESIPT process in the S1 state. However, the other excited-state dynamical results are strongly related to the number of beads. At 50 and 100 K, the nuclear quantum effects (NQEs) are very important; therefore, the excited-state dynamical results change significantly with the bead number. For example, the S1 decay time deduced from time-dependent state populations becomes longer as the bead number increases. Nevertheless, an essentially convergent trend is observed when the bead number is close to 10. In contrast, at 300 K, the NQEs become weaker and the above dynamical results converge very quickly even with 1 bead. Most importantly, the NQEs seriously affect the excited-state decay mechanism of o-HBI. At 50 and 100 K, most trajectories decay to the S0 state via perpendicular keto MECIs, whereas, at 300 K, only twisted keto MECIs are responsible for the excited-state decay. The present work not only comprehensively explores the temperature-dependent photoinduced dynamics of o-HBI, but also demonstrates the importance and necessity of NQEs in nonadiabatic dynamics simulations, especially at relatively low temperatures.

Modifying Fluorescent Protein Chromophore to Detect Protamine and Application in Real Serum Samples

AbstractAs effective methods to accurately and rapidly detect protamine remains a major challenge, here we present a fluorescent protein chromophore‐based probe (IDL‐FP) utilizing twisted‐intramolecular charge transfer (TICT) to detect protamine. The introduction of an electron‐donor group 4‐dimethylaminobenzaldehyde extends the emission wavelength of IDL‐FP to the red region. The deprotection of t‐butyloxy carbonyl groups can then generate an electronegative carboxyl group, which promotes the binding to protamine via electrostatic attraction. Thus, IDL‐FP is sensitive and specific towards protamine and can serve as a highly selective detector for protamine with a detection limit as low as 8.87 ng/mL. As IDL‐FP exhibited good stability in physiological pH environment, we successfully apply IDL‐FP to detect spiked‐in protamine in human serum. In summary, probe IDL‐FP is a promising tool for the quantitative determination of protamine.

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.

What Happens to a Pyrrole Hemithioindigo Photoswitch Trapped in a Fluorescent Protein?

Artificial molecular photoswitches that can be reversibly controlled into different configurations by external light stimulation have broad application prospects in various fields, such as materials and chemical biology. Among them, the pyrrole hemithioindigo (PHT) photoswitch has a geometric structure similar to that of the fluorescent protein chromophore. What happens when the chromophore is replaced by PHT, and does it achieve similar functions to the original one? To answer these questions, we carried out ONIOM(QM/MM) and classical molecular dynamics studies on the photoisomerization mechanism and spectroscopic properties of PHT in the fluorescent protein. The results showed that in the protein environment, the fate of excited PHT is governed by the competition between fluorescence emission and hula-twist isomerization. Due to the strong steric hindrance effects caused by the interlacing residues in the protein that restrict the PHT conformation transformation, the cis-to-trans isomerization process of PHT needs to overcome a barrier of at least 4.9 kcal/mol; thus, fluorescence emission is more dominant in competition. It is also found that the intermolecular interaction between LYS67 and the carbonyl oxygen of PHT has a significant effect on the fluorescence emission. These results revealed the photochemical reaction mechanism of a light-driven molecular switch in the fluorescent protein and provided further theoretical support for the field of chemical biology.

Regioselective and solvent-dependent photoisomerization induced internal conversion in red fluorescent protein chromophore analogues.

Photophysical properties of three red fluorescent protein (RFP) chromophore analogues are reported here. The three RFP chromophore analogues differ in the additional conjugation present in the RFP chromophore. The three chromophores do not exhibit any solvent effect in both absorption and fluorescence spectra. The photoirradiation experiments and recording of 1H NMR before and after irradiation on one of the three RFP model chromophores show isomerization of the (Z,E) diastereomer to the (E,E) diastereomer. Calculation of S0 and S1 potential energy curves shows the preference for isomerization through the exocyclic C[double bond, length as m-dash]C bond with Z-stereochemistry, thus corroborating the experimental results. The computational studies also suggest that torsional motion along the exocyclic C[double bond, length as m-dash]C bond pushes the molecules to a conical intersection, thus paving the pathway for radiationless deactivation.

Biodegradable fluorescent protein chromophore nanoparticles for hypoxic two-photon photodynamic therapy.

In this paper, biodegradable red fluorescent protein (RFP) chromophore analogue DPFP-SS-FA nanoparticles were synthesized for hypoxic two-photon photodynamic therapy. The maximum emission wavelength of DPFP-SS-FA is in the red-to-near-infrared region at 674 nm. Interestingly, these DPFP-SS-FA nanoparticles remain stable under physiological conditions, but deplete glutathione and disintegrate into the RFP chromophore analogue monomer in the tumor microenvironment. Meanwhile, electron paramagnetic resonance data have shown that DPFP-SS-FA produced enhanced 1O2/O2˙- signals after glutathione depletion causing an enhanced PDT effect. DPFP-SS-FA has negligible cell dark toxicity and high phototoxicity in hypoxic environments, indicating the outstanding hypoxia-overcoming ability of DPFP-SS-FA. In addition, due to its folic acid receptor and lysosome dual-targeting ability, DPFP-SS-FA is highly enriched in A-549 tumor cells. In particular, the hypoxic two-photon photodynamic therapy mediated by DPFP-SS-FA nanoparticles was validated in a zebrafish tumor model. Under 800 nm two-photon excitation, DPFP-SS-FA enabled bright two-photon fluorescence imaging and significantly inhibited the growth of tumor cells in zebrafish. The biodegradable DPFP-SS-FA nanoparticles reasonably constructed in this study can serve as excellent candidates for efficient hypoxic two-photon photosensitizers to treat deep tumor tissues.

Fluorescent protein chromophores modified with aromatic heterocycles for photodynamic therapy and two-photon fluorescence imaging.

In this paper, three fluorescent protein chromophore analogs PFPAr (PFPP, PFPC, and PFPT) were synthesized and proved to be useful for photodynamic therapy and two-photon fluorescence imaging. By adding five- or six-membered aromatic heterocycles to the photosensitizer PFP, we obtained three fluorescent protein photosensitizers PFPAr with better performances. As a demonstration, compared with the reported photosensitizer PFP, photosensitizer PFPP exhibits larger emission wavelengths (701 nm) and achieves a slight enhancement in the efficiency of singlet oxygen (ΦΔ = 23%). Notably, PFPP can perform good two-photon fluorescence imaging with an 800 nm femtosecond laser in zebrafish. In in vitro cytotoxicity assays, PFPP shows good phototoxicity (IC50 = 4.12 μM) and acceptable dark toxicity (cell viability assay >90%). The reactive oxygen imaging experiments and AO/EB double staining assay indicate that PFPP can generate singlet oxygen to eliminate A-549 tumor cells effectively with photoexcitation of 460 nm blue light (20 mW cm-2). Furthermore, PFPP can label the lysosomes of tumor cells with high specificity for lysosomes (Pearson's correlation coefficient of 0.91). Thus, our study demonstrated that the rational introduction of aromatic heterocycles into fluorescent protein photosensitizers can effectively enhance the key parameters of photosensitivity and pave the way for further two-photon photodynamic therapy.

New-generation electron-propagator methods for vertical electron detachment energies of molecular anions: benchmarks and applications to model green-fluorescent-protein chromophores.

Ab initio electron-propagator calculations continue to be useful companions to experimental investigations of electronic structure in molecular anions. A new generation of electron-propagator methods recently has surpassed its antecedents' predictive accuracy and computational efficiency. Interpretive clarity has been conserved, for no adjustable parameters have been introduced in the preparation of molecular orbitals or in the formulation of approximate self-energies. These methods have employed the diagonal self-energy approximation wherein each Dyson orbital equals a canonical Hartree-Fock orbital times the square root of a probability factor. Numerical tests indicate that explicitly renormalized, diagonal self-energies are needed when Dyson orbitals have large valence nitrogen, oxygen or fluorine components. They also demonstrate that even greater accuracy can be realized with generalizations that do not employ the diagonal self-energy approximation in the canonical Hartree-Fock basis. Whereas the diagonal methods have fifth-power arithmetic scaling factors, the non-diagonal generalizations introduce only non-iterative sixth-power contractions. Composite models conserve the accuracy of the most demanding combinations of self-energy approximations and flexible basis sets with drastically reduced computational effort. Composite-model results on anions that resemble the chromophore of the green fluorescent protein illustrate the interpretive capabilities of explicitly renormalized self-energies. Accurate predictions on the lowest vertical electron detachment energy of each anion confirm experimental data and the utility of the diagonal self-energy approximation.

Green fluorescent protein chromophore-based covalent organic polymers (GFPC-COPs): sensing of nitroaromatic organic pollutants and explosives

Fluorescent covalent organic polymers based on green fluorescent protein chromophores serve as excellent sensory materials for sensing of nitroaromatic compounds containing acidic hydrogens, e.g., nitrophenols, nitrothiophenols and nitroanilines.

OpenMM 8: Molecular Dynamics Simulation with Machine Learning Potentials.

Machine learning plays an important and growing role in molecular simulation. The newest version of the OpenMM molecular dynamics toolkit introduces new features to support the use of machine learning potentials. Arbitrary PyTorch models can be added to a simulation and used to compute forces and energy. A higher-level interface allows users to easily model their molecules of interest with general purpose, pretrained potential functions. A collection of optimized CUDA kernels and custom PyTorch operations greatly improves the speed of simulations. We demonstrate these features in simulations of cyclin-dependent kinase 8 (CDK8) and the green fluorescent protein chromophore in water. Taken together, these features make it practical to use machine learning to improve the accuracy of simulations with only a modest increase in cost.