Fluorogenic Protein Labeling Research Articles

Ethynylbenzaldehydes as novel reaction-based “turn-on” fluorescent probes for primary amine detection in solution, vapor, food, proteins, and live cells

Amine detection is of great importance as excess volatile amine exposure and increased concentration of biogenic amines in human body can cause adverse effects to human health. Thus, the development of easy-accessible and efficient fluorescent probes for amine detection is of great importance. In this study, we are the first to establish a cascade reaction sequence featuring imine formation followed by 6-endo-dig cyclization reaction for the development of sensitive and selective “turn-on” fluorescent probes toward primary amines by using the ortho-ethynylbenzaldehydes (EBAs) as the reaction site. The corresponding isoquinolinium products generated give a significant red-shifted absorbance change and highly intense “turn-on” fluorescence. Nine EBAs (EBA-1a-1e, EBA-2–5) were designed and synthesized to study the structure-photophysical property relationship (SPPR) for selective primary amine sensing. Our EBAs possessed a highly sensitive response toward primary amines in aqueous conditions within 30 min (LOD as low as 0.45 µM) with tunable absorption (338–430 nm) and emission (410–535 nm), large Stokes shift (up to 115 nm), and high fluorescence quantum yield (up to 0.86). EBA-loaded paper strips were prepared as a portable low cost tool for amine detection in solution, vapor, and food. Fluorogenic protein labelling and in-gel fluorescent imaging were efficiently achieved by using EBAs in which the fluorescence signal was obviously observed by naked eyes under 365 nm UV lamp. Moreover, the favorable membrane permeabilization, low to moderate cytotoxicity, and “turn-on” fluorescent response of EBA-1a-1e and EBA-4–5 allow wash-free live cell imaging. Interestingly, EBA-1a-1e and EBA-4 showed high selectivity to fluorescent imaging of mitochondria.

A general strategy for in situ assembly of light-up fluorophores via bioorthogonal Suzuki-Miyaura cross-coupling

Herein we presented a general strategy for in situ assembly of intramolecular charge-transfer (ICT)-based light-up fluorophores via bioorthogonal Suzuki-Miyaura cross-coupling reaction. By introducing iodo group at the appropriate position, five fluorophores with different scaffolds including naphthalimide, coumarin, naphthalene sulfonate, nitrobenzoxadiazole, and acetonaphthone, were designed as bioorthogonal multicolor fluorogenic probes, which could produce significant fluorescence enhancement and high fluorescence quantum yield after Suzuki-Miyaura reaction with aryl boronic acid or boronate. Manipulating the substituents and π scaffold in the fluorophores allows fine-tuning of their photophysical properties. With this strategy, we succeeded in peptide conjugation, no-wash fluorogenic protein labeling, and mitochondria-selective bioorthogonal imaging in live cells.

A base-controlled switch of SO2 reincorporation in photocatalyzed radical difunctionalization of alkenes

A base-controlled switch of SO2 reincorporation in photocatalyzed radical difunctionalization of alkenes

Suzuki Cross‐Coupling Reaction with Genetically Encoded Fluorosulfates for Fluorogenic Protein Labeling

A palladium-catalyzed cross-coupling reaction with aryl halide functionalities has recently emerged as a valuable tool for protein modification. Herein, a new fluorogenic modification methodology for proteins, with genetically encoded fluorosulfate-l-tyrosine, which exhibits high efficiency and biocompatibility in bacterial cells as well as in aqueous medium, is described. Furthermore, the cross-coupling of 4-cyanophenylboronic acid on green fluorescent protein was shown to possess a unique fluorogenic property, which could open up the possibility of a responsive "off/on" switch with great potential to enable spectroscopic imaging of proteins with minimal background noise. Taken together, a convenient and efficient catalytic system has been developed that may provide broad utilities in protein visualization and live-cell imaging.

A Visible and Near-Infrared Light Activatable Diazocoumarin Probe for Fluorogenic Protein Labeling in Living Cells.

Chemical modification of proteins in living cells permits valuable glimpses into the molecular interactions that underpin dynamic cellular events. While genetic engineering methods are often preferred, selective labeling of endogenous proteins in a complex intracellular milieu with chemical approaches represents a significant challenge. In this study, we report novel diazocoumarin compounds that can be photoactivated by visible (430-490 nm) and near-infrared light (800 nm) irradiation to photo-uncage reactive carbene intermediates, which could subsequently undergo an insertion reaction with concomitant fluorescence "turned on". With these new molecules in hand, we have developed a new approach for rapid, selective, and fluorogenic labeling of endogenous protein in living cells. By using CA-II and eDHFR as model proteins, we demonstrated that subcellular localization of proteins can be precisely visualized by live-cell imaging and protein levels can be reliably quantified in multiple cell types using flow cytometry. Dynamic protein regulations such as hypoxia-induced CA-IX accumulation can also be detected. In addition, by two-photon excitation with an 800 nm laser, cell-selective labeling can also be achieved with spatially controlled irradiation. Our method circumvents the cytotoxicity of UV light and obviates the need for introducing external reporters with "click chemistries". We believe that this approach of fluorescence labeling of endogenous protein by bioorthogonal photoirradiation opens up exciting opportunities for discoveries and mechanistic interrogation in chemical biology.

Visualizing GABA A Receptor Trafficking Dynamics with Fluorogenic Protein Labeling

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAARs), exhibit highly dynamic trafficking and cell surface mobility. Regulated trafficking to and from the surface is a critical determinant of GABAAR neurotransmission. Receptors delivered by exocytosis diffuse laterally in the plasma membrane, with tethering and reduced movement at synapses occurring through receptor interactions with the subsynaptic scaffold. After diffusion away from synapses, receptors are internalized by clathrin-dependent endocytosis at extrasynaptic sites and can be either recycled back to the cell membrane or degraded in lysosomes. To study the dynamics of these key trafficking events in neurons, we have developed novel optical methods based around receptors containing a dual-tagged γ2 subunit (γ2pHFAP) in combination with fluorogen dyes. Specifically, the GABAAR γ2 subunit is tagged with a pH-sensitive green fluorescent protein and a fluorogen-activating peptide (FAP). The FAP allows receptor labeling with fluorogen dyes that are optically silent until bound to the FAP. Combining FAP and fluorescent imaging with organelle labeling allows novel and accurate measurement of receptor turnover and accumulation into intracellular compartments under basal conditions in scenarios ranging from in vitro seizure models to drug exposure paradigms. Here we provide a protocol to track and quantify receptors in transit from the neuronal surface to endosomes and lysosomes. This protocol is readily applicable to cell lines and primary cells, allowing rapid quantitative measurements of receptor surface levels and postendocytic trafficking decisions. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Preparation of cortical neuronal cultures for imaging assays Basic Protocol 2: Surface receptor internalization and trafficking to early endosomes Basic Protocol 3: Measurement of receptor steady state surface level, synaptic level, and lysosomal targeting.

Development of an effective protein-labeling system based on smart fluorogenic probes

Proteins are an important component of living systems and play a crucial role in various physiological functions. Fluorescence imaging of proteins is a powerful tool for monitoring protein dynamics. Fluorescent protein (FP)-based labeling methods are frequently used to monitor the movement and interaction of cellular proteins. However, alternative methods have also been developed that allow the use of synthetic fluorescent probes to target a protein of interest (POI). Synthetic fluorescent probes have various advantages over FP-based labeling methods. They are smaller in size than the fluorescent proteins, offer a wide variety of colors and have improved photochemical properties. There are various chemical recognition-based labeling techniques that can be used for labeling a POI with a synthetic probe. In this review, we focus on the development of protein-labeling systems, particularly the SNAP-tag, BL-tag, and PYP-tag systems, and understanding the fluorescence behavior of the fluorescently labeled target protein in these systems. We also discuss the smart fluorogenic probes for these protein-labeling systems and their applications. The fluorogenic protein labeling will be a useful tool to investigate complex biological phenomena in future work on cell biology.

Development of 4-oxime-1,8-naphthalimide as a bioorthogonal turn-on probe for fluorogenic protein labeling.

4-Oxime-1,8-naphthalimide was reported as a novel bioorthogonal turn-on probe based on 1,3-dipolar cycloaddition reactions between in situ generated nitrile oxide and alkenes/alkynes. The resulting isoxazole products displayed dramatically strong fluorescence enhancement upon photoirradiation through isoxazole-oxazole photoisomerization. This new methodology was successfully applied for in situ fluorogenic protein labeling.

Rational design of a highly reactive dicysteine peptide tag for fluorogenic protein labelling.

Rationally designed libraries of a short helical peptide sequence containing two cysteine residues were screened kinetically for their reactivity towards complementary dimaleimide fluorogens. This screening revealed variant sequences whose reactivity has been increased by an order of magnitude relative to the original sequence. The most reactive engineered sequences feature mutant residues bearing positive charges, suggesting the pKa values of the adjacent thiol groups have been significantly lowered, through electrostatic stabilization of the thiolate ionization state. pH-Rate profiles measured for several mutant sequences support this mechanism of rate enhancement. The practical utility of the enhanced reactivity of the final engineered dicysteine tag ('dC10*') was then demonstrated in the fluorogenic intracellular labelling of histone H2B in living HeLa cells.

Fluorogenic Protein Labeling Probes to Study the Morphological Interplay between PreLamin and Mature Lamin

Although many protein labeling probes have been developed to elucidate the trafficking and turnover processes of cell surface proteins, real-time tracking of intracellular proteins remains a challenging task. Herein, we describe a new design to construct a cell-permeable, photostable, and far-red fluorescent turn-on probe to enable no-wash, organelle-specific, and long-term visualization of intracellular SNAP-tagged proteins in living cells. When the probe was used in dual-color pulse chase labeling experiments to differentiate between preLamin and mature Lamin, our results reveal that the shape of mature Lamin can be altered by the newly synthesized preLamin and that this alteration is progressive, cumulative, and due to a concentration-dependent dominant-negative effect of preLamin. We believe that this probe can also be applied to other intracellular proteins whose cellular localization and synthesis changes dynamically in response to external stimuli.

Improved Photoinduced Fluorogenic Alkene-Tetrazole Reaction for Protein Labeling.

The 1,3-dipolar cycloaddition reaction between an alkene and a tetrazole represents one elegant and rare example of fluorophore-forming bioorthogonal chemistry. This is an attractive reaction for imaging applications in live cells that requires less intensive washing steps and/or needs spatiotemporal resolutions. In the present work, as an effort to improve the fluorogenic property of the alkene-tetrazole reaction, an aromatic alkene (styrene) was investigated as the dipolarophile. Over 30-fold improvement in quantum yield of the reaction product was achieved in aqueous solution. According to our mechanistic studies, the observed improvement is likely due to an insufficient protonation of the styrene-tetrazole reaction product. This finding provides useful guidance to the future design of alkene-tetrazole reactions for biological studies. Fluorogenic protein labeling using the styrene-tetrazole reaction was demonstrated both in vitro and in vivo. This was realized by the genetic incorporation of an unnatural amino acid containing the styrene moiety. It is anticipated that the combination of styrene with different tetrazole derivatives can generally improve and broaden the application of alkene-tetrazole chemistry in real-time imaging in live cells.

Development of cyanine probes with dinitrobenzene quencher for rapid fluorogenic protein labelling.

A multicolour protein labelling technique using a protein tag and fluorogenic probes is a powerful approach for spatio-temporal analyses of proteins in living cells. Since cyanine fluorophores have attractive properties for multicolour imaging of proteins, there is a huge demand to develop fluorogenic cyanine probes for specific protein labelling in living cells. Herein, we develop fluorogenic cyanine probes for labelling a protein tag by using a dinitrobenzene fluorescence quencher. The probes enhanced fluorescence intensity upon labelling reactions and emitted orange or far-red fluorescence. Intramolecular interactions between the cyanine fluorophores and the dinitrobenzene quencher led not only to fluorescence quenching of the probes in the free state but also to promotion of labelling reactions. Furthermore, the probes successfully imaged cell-surface proteins without a washing process. These findings offer valuable information on the design of fluorogenic cyanine probes and indicate that the probes are useful as novel live-cell imaging tools.This article is part of the themed issue 'Challenges for chemistry in molecular imaging'.

Fluorogenic protein labeling using a genetically encoded unstrained alkene.

We developed a new fluorogenic bioorthogonal reaction that is based on the inverse electron-demand Diels-Alder reaction between styrene (an unstrained alkene) and a simple tetrazine. The reaction forms a new fluorophore with no literature precedent. We have identified an aminoacyl-tRNA synthetase/tRNA pair for the efficient and site-specific incorporation of a styrene-containing amino acid into proteins in response to amber nonsense codon. Fluorogenic labeling of purified proteins and intact proteins in live cells were demonstrated. The fluorogenicity of the styrene-tetrazine reaction can be potentially applied to the study of protein folding and function under physiological conditions with low background fluorescence interference.

Fluorogenic protein labelling: a review of photophysical quench mechanisms and principles of fluorogen design

Fluorescent labelling of specific proteins in complex biological systems remains an important challenge in chemical biology. One promising approach comprises the use of small molecules designed to react specifically with a targeted protein of interest and to increase in fluorescent intensity following this reaction. This kind of fluorogenic reaction generally derives from fluorescence quenching in the unreacted probe that is abrogated over the course of the reaction. Herein, we review the mechanistic principles of three major photophysical quenching mechanisms involving Förster resonance energy transfer (FRET), through-bond energy transfer (TBET), and photoinduced electron transfer (PeT). We then present design principles for novel fluorogenic probes based on an understanding of these quench mechanisms, with emphasis on the emerging utility of density functional theory (DFT) calculations in the design process.

Small-Molecule-Based Protein-Labeling Technology in Live Cell Studies: Probe-Design Concepts and Applications

The use of genetic engineering techniques allows researchers to combine functional proteins with fluorescent proteins (FPs) to produce fusion proteins that can be visualized in living cells, tissues, and animals. However, several limitations of FPs, such as slow maturation kinetics or issues with photostability under laser illumination, have led researchers to examine new technologies beyond FP-based imaging. Recently, new protein-labeling technologies using protein/peptide tags and tag-specific probes have attracted increasing attention. Although several protein-labeling systems are com mercially available, researchers continue to work on addressing some of the limitations of this technology. To reduce the level of background fluorescence from unlabeled probes, researchers have pursued fluorogenic labeling, in which the labeling probes do not fluoresce until the target proteins are labeled. In this Account, we review two different fluorogenic protein-labeling systems that we have recently developed. First we give a brief history of protein labeling technologies and describe the challenges involved in protein labeling. In the second section, we discuss a fluorogenic labeling system based on a noncatalytic mutant of β-lactamase, which forms specific covalent bonds with β-lactam antibiotics such as ampicillin or cephalosporin. Based on fluorescence (or Förster) resonance energy transfer and other physicochemical principles, we have developed several types of fluorogenic labeling probes. To extend the utility of this labeling system, we took advantage of a hydrophobic β-lactam prodrug structure to achieve intracellular protein labeling. We also describe a small protein tag, photoactive yellow protein (PYP)-tag, and its probes. By utilizing a quenching mechanism based on close intramolecular contact, we incorporated a turn-on switch into the probes for fluorogenic protein labeling. One of these probes allowed us to rapidly image a protein while avoiding washout. In the future, we expect that protein-labeling systems with finely designed probes will lead to novel methodologies that allow researchers to image biomolecules and to perturb protein functions.

Protein labeling with fluorogenic probes for no-wash live-cell imaging of proteins

Protein labeling by using a protein tag and its specific fluorescent probe is increasingly becoming a useful technique for the real-time imaging of proteins in living cells. Recently, fluorogenic probes for protein labeling were developed. When using these probes, a washing step is not required for the removal of free probes from the cells, thus, allowing rapid detection of proteins in living cells with high signal-to-noise ratio. Various chemical principles have been applied in the designing of probes to include a turn-on fluorescence switch that is activated by the protein labeling reaction. In this review, we describe about the design strategy of the probes and the advances in fluorogenic protein labeling systems.

Inside Cover: Fluorogenic Protein Labeling through Photoinduced Electron Transfer-Based BL-Tag Technology (Chem. Asian J. 2/2012)

Inside Cover: Fluorogenic Protein Labeling through Photoinduced Electron Transfer-Based BL-Tag Technology (Chem. Asian J. 2/2012)

Fluorogenic Protein Labeling through Photoinduced Electron Transfer-Based BL-Tag Technology

Keeping quenchers on a short leash: Shortening of the linker chain length combined with modification of the quencher moiety was found to improve fluorogenic probes useful for protein labeling by the mutant β-lactamase-tag (BL-tag) technology. The most effective probe, CC3 DNB, displays fluorescence quenching by photoinduced electron transfer (PET) and results in fluorescent protein labeling with fast kinetics.

De novo helical peptides as target sequences for a specific, fluorogenic protein labelling strategy

New methods are needed to selectively label proteins in a manner that minimally perturbs their structures and functions. We have developed a 'small molecule'-based labelling technique that relies on the use of dimaleimide fluorogens that react with a target peptide sequence that presents appropriately spaced, solvent-exposed Cys residues. The thiol addition reaction between target sequence and dimaleimide fluorogen restores the latent fluorescence of the latter and results in the covalent fluorescent labelling of the protein of interest (J. Guy, K. Caron, S. Dufresne, S. W. Michnick, W. G. Skene and J. W. Keillor, J. Am. Chem. Soc., 2007, 129, 11969-11977). We demonstrated the proof-of-principle of this method previously, using a dicysteine mutant of the helical protein Fos (S. Girouard, M.-H. Houle, A. Grandbois, J. W. Keillor and S. W. Michnick, J. Am. Chem. Soc., 2005, 127, 559-566). Herein, we present the design of a novel peptide sequence presenting two Cys residues separated by two turns of an alpha-helix. The secondary structure of this sequence was confirmed by CD spectroscopy, before and after the fluorescent labelling reaction. A new series of di(3-methylmaleimide) fluorogens was prepared and kinetically evaluated, tuning their reactivity toward the target sequence. Attempts were made to increase the reactivity of the parent target sequence by rational design; however, the introduction of basic His residues in the vicinity of one or more Cys residues did not have the desired effect. Finally, epidermal growth factor receptors bearing the de novo target sequence were specifically labelled with a di(3-methylmaleimide) fluorescein fluorogen, validating our method for specific cell-surface labelling of proteins. A wide variety of fluorogen and peptide designs can be envisioned with potential applications to multiplexed labelling for the study of temporal and spatial dynamics of protein expression.

Convergent Preparation and Photophysical Characterization of Dimaleimide Dansyl Fluorogens: Elucidation of the Maleimide Fluorescence Quenching Mechanism

Dimaleimide fluorogens are being developed for application to fluorescent protein labeling. In this method, fluorophores bearing two maleimide quenching groups do not fluoresce until both maleimide groups have undergone thiol addition reactions with the Cys residues of the target protein sequence [J. Am. Chem. Soc. 2005, 127, 559-566]. In this work, a new convergent synthetic route was developed that would allow any fluorophore to be attached via a linker to a dimaleimide moiety in a modular fashion. Series of dimaleimide and dansyl derivatives were thus prepared conveniently and used to elucidate the mechanism of maleimide quenching. Intersystem crossing was ruled out as a potential quenching pathway, based on the absence of a detectable triplet intermediate by laser flash photolysis. Stern-Volmer rate constants were measured with exogenous dimaleimide quenchers and found to be close to the diffusion-controlled limits, consistent with electron transfer being thermodynamically favorable. The thermodynamic feasibility of the photoinduced electron transfer (PET) quenching mechanism was verified by cyclic voltammetry. The redox potentials measured for dansyl and maleimide confirm that electron transfer from the dansyl excited state to a pendant maleimide group is exergonic and is responsible for fluorescence quenching of the fluorogens studied herein. Taking this PET quenching mechanism into account, future fluorogenic protein labeling agents will be designed with spacers of variable length and rigidity to probe the structure-property PET efficiency relationship.