Green Fluorescent Protein Chromophore Research Articles

GFP chromophore derived amphiphiles containing [12]aneN3 and biotin moieties as bio-imaging and targeting nonviral gene vectors

GFP chromophore derived amphiphiles containing [12]aneN3 and biotin moieties as bio-imaging and targeting nonviral gene vectors

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.

General Installation of (4H)-Imidazolone cis-Amide Bioisosteres Along the Peptide Backbone.

Imidazolones represent an important class of heterocycles present in a wide range of pharmaceuticals, metabolites, and bioactive natural products and serve as the active chromophore in green fluorescent protein. Recently, imidazolones have received attention for their ability to act as a nonaromatic amide bond bioisotere which improves pharmacological properties. Herein, we present a tandem amidine installation and cyclization with an adjacent ester to yield (4H)-imidazolone products. Using amino acid building blocks, we can access the first examples of α-chiral imidazolones that have been previously inaccessible. Additionally, our method is amenable to on-resin installation which can be seamlessly integrated into existing solid-phase peptide synthesis protocols. Finally, we show that peptide imidazolones are potent cis-amide bond surrogates that preorganize linear peptides for head-to-tail macrocyclization. This work represents the first general approach to the backbone and side-chain insertion of imidazolone bioisosteres at various positions in linear and cyclic peptides.

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

Understanding 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.

A Molecular Hybrid of the GFP Chromophore and 2,2'-Bipyridine: An Accessible Sensor for Zn2+ Detection with Fluorescence Microscopy.

The few commercially available chemosensors and published probes for in vitro Zn2+ detection in two-photon microscopy are compromised by their flawed spectroscopic properties, causing issues in selectivity or challenging multistep syntheses. Herein, we present the development of an effective small molecular GFP chromophore-based fluorescent chemosensor with a 2,2'-bipyridine chelator moiety (GFZnP BIPY) for Zn2+ detection that has straightforward synthesis and uncompromised properties. Detailed experimental characterizations of the free and the zinc-bound compounds within the physiologically relevant pH range are presented. Excellent photophysical characteristics are reported, including a 53-fold fluorescence enhancement with excitation and emission maxima at 422 nm and 492 nm, respectively. A high two-photon cross section of 3.0 GM at 840 nm as well as excellent metal ion selectivity are reported. In vitro experiments on HEK 293 cell culture were carried out using two-photon microscopy to demonstrate the applicability of the novel sensor for zinc bioimaging.

Quantum dynamics of excited state proton transfer in green fluorescent protein.

Photoexcitation of green fluorescent protein (GFP) triggers long-range proton transfer along a "wire" of neighboring protein residues, which, in turn, activates its characteristic green fluorescence. The GFP proton wire is one of the simplest, most well-characterized models of biological proton transfer but remains challenging to simulate due to the sensitivity of its energetics to the surrounding protein conformation and the possibility of non-classical behavior associated with the movement of lightweight protons. Using a direct dynamics variational multiconfigurational Gaussian wavepacket method to provide a fully quantum description of both electrons and nuclei, we explore the mechanism of excited state proton transfer in a high-dimensional model of the GFP chromophore cluster over the first two picoseconds following excitation. During our simulation, we observe the sequential starts of two of the three proton transfers along the wire, confirming the predictions of previous studies that the overall process starts from the end of the wire furthest from the fluorescent chromophore and proceeds in a concerted but asynchronous manner. Furthermore, by comparing the full quantum dynamics to a set of classical trajectories, we provide unambiguous evidence that tunneling plays a critical role in facilitating the leading proton transfer.

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.

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.

Self-assembly solid-state enhanced fluorescence emission of GFP chromophore analogues: Formation of microsheets and microtubes oriented by molecular skeleton

The p-, m- and o-N,N-dimethylamino analogs of the green fluorescent protein (GFP) chromophore (denoted as p-DBHI, m-DBHI and o-DBHI) were synthesized by 2,3-cycloaddition. These three compounds were structurally characterized by NMR, HRMS and single crystal X-ray diffraction and were shown to be in the Z-form in both the solid phase and solution. Their fluorescence properties and self-assembly behaviors were investigated by UV–Vis, photoluminescence spectroscopy, fluorescence microscopy and scanning electron microscopy. They exhibited low fluorescence quantum yields in both protic and aprotic solvents, which was consistent with the reported results, and strong emissions in the solid state, thus exhibiting aggregation-induced emission (AIE) behaviors. By a solvent exchange method, the p-DBHI and o-DBHI were assembled into microsheets, while the m-DBHI was assembled into microtubule-like structures. The photoluminescence properties of the assemblies were compared with those of the pristine microcrystalline powders obtained by evaporation from organic solvents. The fluorescence quantum yields of the microcrystals obtained by self-assembly were recorded to 9.86 %, 3.37 % and 31.65 %, respectively, which were much higher than those of the corresponding pristine powders (4.71 %, 2.51 % and 17.03 %). This indicated that the fluorescence properties in the solid state depended on the morphologies of the particles.

Two-photon fluorescent chemosensors based on the GFP-chromophore for the detection of Zn2+ in biological samples – From design to application

A library of twelve novel fluorescent GFP chromophore based chemosensors (GFZnPs) was prepared for the detection of Zn2+ in biological samples with two-photon microscopy. The presented compounds are the hybrids of the Zn2+ chelator 8-aminoquinoline motif and the GFP chromophore, exploiting and enhancing the properties of the natural chromophore. In the presented probes, the quenching rotation around the benzylidene carbon of the GFP chromophore is conditionally blocked upon Zn2+ binding, resulting in a fluorescent response to the ion. GFZnPs have an efficient and simple synthesis making them one of the most easily accessible two-photon (2P) Zn2+ sensors. We present the comprehensive spectroscopical characterization of the probe family highlighting their bright fluorescent emission (ε × Φ > 200) at λem∼520 nm upon excitation by λex∼450 nm light. The sensors feature high selectivity for Zn2+ with an affinity in the nanomolar range, excellent water solubility, and usability at a wide pH-range (pH > 6). The relationship between the structure of compounds and their properties was studied and rationalized by theoretical considerations. The 2P-action cross section of the probes reaches δ × Φcomp > 5 GM at λex,2P= 900 nm, with an increase up to 200-fold, which makes them one of the best two-photon probes for Zn2+. The applicability of selected probes was demonstrated by epifluorescent and 2P microscopy on cultured cells and brain slices. This work expands the toolbox of efficient two-photon zinc sensors with a valuable new family.

Oxidized Low-Density Lipoprotein (Ox-LDL)-Triggered Double-Lock Probe for Spatiotemporal Lipoprotein Oxidation and Atherosclerotic Plaque Imaging.

Low-density lipoprotein (LDL), especially oxidative modified LDL (Ox-LDL), is the key risk factor for plaque accumulation and the development of cardiovascular disease. Herein, a highly specific Ox-LDL-triggered fluorogenic-colorimetric probe Pro-P1 is developed for visualizing the oxidation and aggregation progress of lipoproteins and plaque. A series of green fluorescent protein chromophores with modified donor-acceptor structures, containing carbazole as an electron donor and various substituents including pyridine-vinyl (P1), phenol-vinyl (P2), N, N-dimethylaniline-vinyl (P3), and thiophene-vinyl (P4), have been synthesized and evaluated. Emission spectroscopy and theoretical calculations of P1-P4 indicate that P1 shows enhanced green fluorescence (λem = 560nm) by inhibiting its twisted intramolecular charge transfer in the presence of Ox-LDL. This feature allows the selection of P1 as a sensitive probe to directly visualize ferroptosis and Cu2+ -mediated LDL oxidative aggregation via in situ formation of fluorophore-bound Ox-LDL in living cells. The red-emissive probe Pro-P1 (λem = 660nm) is prepared via borate protection of P1, which can be cleaved into P1 under high expression of HOCl and Ox-LDL condition at the lesion site, resulting in enhanced green emission. The plaque area and size with clear boundaries can be delineated by colorimetric fluorescence imaging and fluorescence lifetime imaging with precise differentiation.

An epoxide-based covalent sensor to detect cardiac proteome aggregation in a cardio-oncology model

Covalent sensors to detect and capture aggregated proteome in stressed cells are rare. Herein, we construct a series of covalent fluorogenic sensors for aggregated proteins by structurally modulating GFP chromophore and arming it with an epoxide warhead. Among them, P2 probe selectively modifies aggregated proteins over folded ones and turns on fluorescence as evidenced by biochemical and mass spectrometry results. The coverage of this epoxide-based covalent chemistry is demonstrated using different types of aggregated proteins. Finally, the covalent fluorescent sensor P2 allows for direct visualization and capture of aggregated proteome in stressed cardiomyocytes and cardiac tissue samples from a cardio-oncology mouse model. The epoxide-based covalent sensor developed herein may become useful for future chemical proteomics analysis of aggregated proteins to dissect the mechanism underlying cardio-oncology.

Elucidating the Role of Hydrogen Bonding in the Optical Spectroscopy of the Solvated Green Fluorescent Protein Chromophore: Using Machine Learning to Establish the Importance of High-Level Electronic Structure.

Hydrogen bonding interactions with chromophores in chemical and biological environments play a key role in determining their electronic absorption and relaxation processes, which are manifested in their linear and multidimensional optical spectra. For chromophores in the condensed phase, the large number of atoms needed to simulate the environment has traditionally prohibited the use of high-level excited-state electronic structure methods. By leveraging transfer learning, we show how to construct machine-learned models to accurately predict the high-level excitation energies of a chromophore in solution from only 400 high-level calculations. We show that when the electronic excitations of the green fluorescent protein chromophore in water are treated using EOM-CCSD embedded in a DFT description of the solvent the optical spectrum is correctly captured and that this improvement arises from correctly treating the coupling of the electronic transition to electric fields, which leads to a larger response upon hydrogen bonding between the chromophore and water.

High-Resolution Spectroscopy and Selective Photoresponse of Cryogenically Cooled Green Fluorescent Protein Chromophore Anions.

By time-resolved action spectroscopy of cryogenically cooled molecular ions, we have achieved a remarkable vibrational resolution in the photoresponse of the deprotonated green fluorescent protein (GFP) chromophore, a key molecular unit in the bioimaging of living cells. We define four characteristic spectral regions of the S0-S1 band with competing electronic and nuclear decay channels. We determine the energy barrier toward internal conversion to be ∼250 cm-1. This inhibits internal conversion and hence statistical fragmentation near the S0-S1 band origin, which is identified at 481.51 ± 0.15 nm (20768 ± 6 cm-1). The origin is red-shifted by only 221 cm-1 compared to that of wild-type GFP at 77 K. This, together with a striking agreement between the vibronic profiles of the protein and its chromophore, suggests their similar photophysics. In combination with theory, the data reveal the coexistence of mutually energy-borrowing mechanisms between nuclei and electrons mediated by specific vibrational modes.

Impact of the Protein Environment on Two-Photon Absorption Cross-Sections of the GFP Chromophore Anion Resolved at the XMCQDPT2 Level of Theory.

The search for fluorescent proteins with large two-photon absorption (TPA) cross-sections and improved brightness is required for their efficient use in bioimaging. Here, we explored the impact of a single-point mutation close to the anionic form of the GFP chromophore on its TPA activity. We considered the lowest-energy transition of EGFP and its modification EGFP T203I. We focused on a methodology for obtaining reliable TPA cross-sections for mutated proteins, based on conformational sampling using molecular dynamics simulations and a high-level XMCQDPT2-based QM/MM approach. We also studied the numerical convergence of the sum-over-states formalism and provide direct evidence for the applicability of the two-level model for calculating TPA cross-sections in EGFP. The calculated values were found to be very sensitive to changes in the permanent dipole moments between the ground and excited states and highly tunable by internal electric field of the protein environment. In the case of the GFP chromophore anion, even a single hydrogen bond was shown to be capable of drastically increasing the TPA cross-section. Such high tunability of the nonlinear photophysical properties of the chromophore anions can be used for the rational design of brighter fluorescent proteins for bioimaging using two-photon laser scanning microscopy.

Meta-CF3-Substituted Analogues of the GFP Chromophore with Remarkable Solvatochromism.

In this work, we have shown that the introduction of a trifluoromethyl group into the me-ta-position of arylidene imidazolones (GFP chromophore core) leads to a dramatic increase in their fluorescence in nonpolar and aprotic media. The presence of a pronounced solvent-dependent gradation of fluorescence intensity makes it possible to use these substances as fluorescent polarity sensors. In particular, we showed that one of the created compounds could be used for selective labeling of the endoplasmic reticulum of living cells.

Cocrystal Approach to Modulate the Photoluminescent Properties of a GFP Chromophore Analogue: Role of Halogen/Hydrogen Bonding in Achieving a Wide Range of Solid-State Fluorescence Emissions

Fine-tuning the photophysical properties of fluorescent organic solids is essential to attain multicolor displays and meet the demand for futuristic light-emitting materials. Here, we report the tunable luminescence of a green fluorescent protein (GFP) chromophore analogue, 3,4,5-TIA (A), based on the formation of two-component molecular cocrystals with six different coformers. Coformers selected to synthesize the binary cocrystals include 1,4-diiodotetrafluorobenzene (B), perfluoronaphthalene (C), 1,4-dibromotetrafluorobenzene (D), 2,3,5,6-tetrafluoroterephthalic acid (E), benzene-1,2,4,5-tetracarbonitrile (F), and benzene-1,2,4,5-tetracarboxylic acid (G). Interestingly, the cocrystals A•C and A•F showed molecular crystal polymorphism with a slight variation in fluorescence, revealing an aggregation-induced emission (AIE). A crystal structure analysis showed the interplay of hydrogen bonding, halogen bonding, and aromatic π-stacking interactions in associating neutral solid components in the cocrystal. All of the novel cocrystals displayed a wide range of photoluminescence ranging from blue to dark orange. The time-dependent density functional theory (TD-DFT) calculations indicate the changes in the energy level structures (HOMO to LUMO) in cocrystals that resulted in variations in fluorescence emission. The study aims to further understand the structure–property relationship between molecular arrangement and photoluminescence.

Fluorescence Decay Analysis of the Model Compounds as an Approach to Photophysical Engineering of Fluorescent Proteins

Studying of structure-function relationships between a chromophore and its protein environment plays a key role in photophysical engineering of fluorescent proteins (FPs), specifically, in the guided designing of their new variants with a higher fluorescence quantum yield (FQY). Known approaches to FQY increasing mostly rely on suppression of the excited state nonradiative processes, but no tools have been suggested for the tuning of the radiative rate constant (kr), which is also a potentially “adjustable” value. Here, we propose an experimental approach in which the synthetic chromophore of FP models the “fixation” of the most important radiationless constants and allows monitoring of the fluorescence lifetime flexibility (as an indicator of the kr value). As a proof-of-concept, we studied the time-resolved fluorescence behavior of the green and blue FP chromophore analogs in diverse chemical environments. The conformationally locked analog of the GFP chromophore in most cases showed monophasic fluorescence decay kinetics with a lifetime of 2.7–3.0 ns, thus adequately modeling the typical behavior of GFPs with the highest FQYs. Under the conditions of stimulated ionization of this chromophore, we observed increased (up to 4.3–4.6 ns) fluorescence lifetimes, which can be interpreted in terms of an increase in the radiative constant (kr). The conformationally locked analog of the Sirius chromophore showed biexponential fluorescence decay kinetics, partly simulating the properties of the blue FPs. In an acetic acid solution, this compound exhibited distinct fluorescent properties (elevated fluorescence intensity with a major lifetime population of ~4 ns), which can be interpreted as the emission of an unusual cationic form of the chromophore.

NanoLuc Luciferase as a Fluorogen-Activating Protein for GFP Chromophore Based Fluorogens.

In this work, we showed that the well-known NanoLuc luciferase can act as a fluorogen activating protein for various arylidene-imidazolones structurally similar to the Kaede protein chromophore. We showed that such compounds can be used as fluorescent sensors for this protein and can also be used in pairs with it in fluorescent microscopy as a genetically encoded tag.

Quantum mechanics/fluctuating charge calculations for absorption spectra of aqueous green fluorescent protein chromophore

AbstractWe applied the quantum mechanics/fluctuating charges (QM/FQ) method to simulate the absorption spectra of three protonated forms of p‐hydroxybenzylidene imidazolone (p‐HBDI) in water. Configurations of solute‐solvent were generated from molecular dynamics (MD) simulation within the context of a hybrid scheme in which solute and solvents were treated at QM and molecular mechanics, respectively. Nonperiodic spherical boundary condition was used. To examine the influence on simulated spectra coming from the geometrical relaxation of solute, the MD simulations were carried out for two cases: frozen and flexible molecular geometry of solute. As a result of our calculations, the simulations using the flexible solute's structure delivered vertical excitation energies closer to experimental data than those of a frozen solute, especially for the neutral case. For spectral broadening, changing from fixed geometry of solute to a flexible one led to a significant increase for both neutral and anionic forms of p‐HBDI, but no effect happened for the cationic one.