Probes For Protein Labeling Research Articles

β-Galactosidase- and Photo-Activatable Fluorescent Probes for Protein Labeling and Super-Resolution STED Microscopy in Living Cells.

We report on the synthesis of two fluorescent probes which can be activated by β-Galactosidase (β-Gal) enzymes and/or light. The probes contained 2-nitro-4-oxybenzyl and 3-nitro-4-oxybenzyl fragments, with β-Gal residues linked to C-4. We performed the enzymatic and photoactivation of the probes in a cuvette and compared them, prior to the labeling of Vimentin-Halo fusion protein in live cells with overexpressed β-galactosidase. The dye fluorescence afforded the observation of enzyme activity by means of confocal and super-resolution optical microscopy based on stimulated emission depletion (STED). The tracing of enzymatic activity with the retention of activated fluorescent products inside cells was combined with super-resolution imaging as a tool for use in biomedicine and life science.

Enabling Universal Access to Rapid and Stable Tetrazine Bioorthogonal Probes through Triazolyl-Tetrazine Formation.

Despite the immense potential of tetrazine bioorthogonal chemistry in biomedical research, the in vivo performance of tetrazine probes is challenged by the inverse correlation between the physiological stability and reactivity of tetrazines. Additionally, the synthesis of functionalized tetrazines is often complex and requires specialized reagents. To overcome these issues, we present a novel tetrazine scaffold-triazolyl-tetrazine-that can be readily synthesized from shelf-stable ethynyl-tetrazines and azides. Triazolyl-tetrazines exhibit improved physiological stability along with high reactivity. We showcase the effectiveness of this approach by creating cell-permeable probes for protein labeling and live cell imaging, as well as efficiently producing 18F-labeled molecular probes for positron emission tomography imaging. By utilizing the readily available pool of functionalized azides, we envisage that this modular approach will provide universal accessibility to tetrazine bioorthogonal tools, facilitating applications in biomedicine and materials science.

Bioisostere-conjugated fluorescent probes for live-cell protein imaging without non-specific organelle accumulation.

Specific labeling of proteins using membrane-permeable fluorescent probes is a powerful technique for bioimaging. Cationic fluorescent dyes with high fluorescence quantum yield, photostability, and water solubility provide highly useful scaffolds for protein-labeling probes. However, cationic probes generally show undesired accumulation in organelles, which causes a false-positive signal in localization analysis. Herein, we report a design strategy for probes that suppress undesired organelle accumulation using a bioisostere for intracellular protein imaging in living cells. Our design allows the protein labeling probes to possess both membrane permeability and suppress non-specific accumulation and has been shown to use several protein labeling systems, such as PYP-tag and Halo tag systems. We further developed a fluorogenic PYP-tag labeling probe for intracellular proteins and used it to visualize multiple localizations of target proteins in the intracellular system. Our strategy offers a versatile design for undesired accumulation-suppressed probes with cationic dye scaffolds and provides a valuable tool for intracellular protein imaging.

Small Plasma Membrane-Targeted Fluorescent Dye for Long-Time Imaging and Protein Degradation Analyses.

Plasma membrane (PM)-targeted fluorescent dyes have become an important tool to visualize morphological and dynamic changes in the cell membrane. However, most of these PM dyes are either too large and thus might potentially perturb the membrane and affect its functions or exhibit a short retention time on the cell membrane. The rapid internalization problem is particularly severe for PM dyes based on cationic and neutral hydrophobic fluorescent dyes, which can be easily transported into the cells by transmembrane potential and passive diffusion mechanisms. In this paper, we report a small but highly specific PM fluorescent dye, PM-1, which exhibits a very long retention time on the plasma membrane with a half-life of approximately 15 h. For biological applications, we demonstrated that PM-1 can be used in combination with protein labeling probes to study ectodomain shedding and endocytosis processes of cell surface proteins and successfully demonstrated that native transmembrane human carbonic anhydrase IX (hCAIX) is degraded via the ectodomain shedding mechanism. In contrast, hCAIX undergoes endocytic degradation in the presence of sheddase inhibitors. We believe that PM-1 can be a versatile tool to provide detailed insights into the dynamic processes of the cell surface proteins.

Upconverting Nanoparticle-Based Photoactive Probes for Highly Efficient Labeling and Isolation of Target Proteins.

Photoaffinity labeling (PAL) has blossomed into a powerful and versatile tool for capture and identification of biomolecular targets. However, low labeling efficiency for specific targets such as lectins, the tedious process for protein purification, inevitable cellular photodamage, and less tissue penetration of UV light are significant challenges. Herein, we reported a near-infrared (NIR) light-driven photoaffinity labeling approach using upconverting nanoparticle (UCNP)-based photoactive probes, which were constructed by assembling photoactive groups and ligands onto NaYF4:Yb,Tm nanoparticles. The novel probes were easily prepared and functionalized, and the labeled proteins can be isolated and purified through simple centrifugation and washing. The advantages of this approach were demonstrated by labeling and isolation of peanut agglutinin (PNA), asialoglycoprotein receptor (ASGPR), and human carbonic anhydrase II (hCAII) from mixed proteins or cell lysates with good selectivity and efficiency, especially for PNA and ASGPR, two lectins that showed low binding affinity to their ligands. More importantly, successful labeling of PNA through pork tissues and ASGPR in mice strongly proved the good tissue penetrating capacity of NIR light and the application potential of UCNP-based photoactive probes for protein labeling in vivo. Biosafety of this approach was experimentally validated by enzyme, cell, and animal work, and we demonstrated that NIR light caused minimal photodamage to enzyme activity compared to UV light, and the UCNP-based photoactive probe presents good biosafety both in vitro and in vivo. We believe that this novel PAL approach will provide a promising tool for study of ligand-protein interactions and identification of biomolecular targets.

Improved Stabilities of Labeling Probes for the Selective Modification of Endogenous Proteins in Living Cells and In Vivo.

To date, various affinity-based protein labeling probes have been developed and applied in biological research to modify endogenous proteins in cell lysates and on the cell surface. However, the reactive groups on the labeling probes are also the cause of probe instability and nonselective labeling in a more complex environment, e. g., intracellular and in vivo. Here, we show that labeling probes composed of a sterically stabilized difluorophenyl pivalate can achieve efficient and selective labeling of endogenous proteins on the cell surface, inside living cells and in vivo. As compared with the existing protein labeling probes, probes with the difluorophenyl pivalate exhibit several advantages, including long-term stability in stock solutions, resistance to enzymatic hydrolysis and can be customized easily with diverse fluorophores and protein ligands. With this probe design, endogenous hypoxia biomarker in living cells and nude mice were successfully labeled and validated by in vivo, ex vivo, and immunohistochemistry imaging.

Rapid and Selective Labeling of Endogenous Transmembrane Proteins in Living Cells with a Difluorophenyl Ester Affinity-Based Probe.

The long-term stability of affinity-based protein labeling probes is crucial to obtain reproducible protein labeling results. However, highly stable probes generally suffer from low protein labeling efficiency and pose significant challenges when labeling low abundance native proteins in living cells. In this paper, we report that protein labeling probes based on an ortho-difluorophenyl ester reactive module exhibit long-term stability in DMSO stock solution and aqueous buffer, yet they can undergo rapid and selective labeling of native proteins. This novel electrophile can be customized with a wide range of different protein ligands and is particularly well-suited for the labeling and imaging of transmembrane proteins. With this probe design, the identity and relative levels of basal and hypoxia-induced transmembrane carbonic anhydrases were revealed by live cell imaging and in-gel fluorescence analysis. We believe that the extension of this difluorophenyl ester reactive module would allow for the specific labeling of various endogenous membrane proteins, facilitating in-depth studies of their distribution and functions in biological processes.

Relationship among structure, cytotoxicity, and Michael acceptor reactivity of quinocidin

Quinocidin (QCD) is a cytotoxic antibiotic with an unusual 3,4-dihydroquinolizinium skeleton. We previously found that QCD captures thiols in neutral aqueous media via a Michael addition-type reaction. However, it remains unclear whether the Michael acceptor reactivity of QCD is responsible for its cytotoxicity. In this study, we synthesized thirteen analogs of QCD to examine the relationship among its structure, cytotoxicity, and reactivity toward thiols. Thiol-trapping experiments and cytotoxicity tests collectively suggested that the Michael acceptor function of QCD is independent of its cytotoxic activity, and that the pyridinium moiety with the hydrophobic side chain is a key structural factor for cytotoxicity. These findings further led us to demonstrate that incorporation of an amide group into the side chain of QCD significantly reduced its toxicity but hardly affected the Michael acceptor function. The present study lays the foundation for QCD-based drug design and highlights the potential of QCD as a unique electrophile for use in the development of covalent inhibitors and protein-labeling probes.

Sydnone-coumarins as clickable turn-on fluorescent sensors for molecular imaging

Copper-catalyzed and copper-free sydnone-alkyne cycloaddition reactions have emerged as complementary click tools for chemical biology but their use in bioorthogonal labeling is still in its infancy. Herein, combinations of alkynes and coumarin-sydnones were screened for their ability to generate pyrazole products displaying strong fluoroscence enhancement compared to reactants. One sydnone was identified as a particularly suitable new turn-on probe for protein labeling.

Quinocidin, a Cytotoxic Antibiotic with an Unusual 3,4-Dihydroquinolizinium Ring and Michael Acceptor Reactivity toward Thiols.

Cytotoxicity-guided fractionation of the culture broth of Actinomadura sp. TP-A0019 led to the isolation of quinocidin (1), a cytotoxic antibiotic with an unusual 3,4-dihydroquinolizinium ring. The structural assignment was made on the basis of high-field NMR experiments and chemical synthesis. Comparison of the spectral properties of 1 with those of its synthetic counterparts revealed that 1 is a racemic mixture of two enantiomers, which showed similar cytotoxicity against HeLa-S3 cells. Nucleophile-trapping experiments demonstrated that 1 captured 2-mercaptoethanol and N-acetyl-l-cysteine by means of a Michael addition-type reaction, but was inert toward 2-aminoethanol and glycolic acid. Notably, the addition of 1 to thiols proceeded smoothly in neutral aqueous media at room temperature. In view of the thiol-trapping ability and the unusual structure, 1 provides a unique scaffold for designing drug leads and protein-labeling probes.

Intracellular Protein-Labeling Probes for Multicolor Single-Molecule Imaging of Immune Receptor-Adaptor Molecular Dynamics.

Single-molecule imaging (SMI) has been widely utilized to investigate biomolecular dynamics and protein-protein interactions in living cells. However, multicolor SMI of intracellular proteins is challenging because of high background signals and other limitations of current fluorescence labeling approaches. To achieve reproducible intracellular SMI, a labeling probe ensuring both efficient membrane permeability and minimal non-specific binding to cell components is essential. We developed near-infrared fluorescent probes for protein labeling that specifically bind to a mutant β-lactamase tag. By structural fine-tuning of cell permeability and minimized non-specific binding, SiRcB4 enabled multicolor SMI in combination with a HaloTag-based red-fluorescent probe. Upon addition of both chemical probes at sub-nanomolar concentrations, single-molecule imaging revealed the dynamics of TLR4 and its adaptor protein, TIRAP, which are involved in the innate immune system. Statistical analysis of the quantitative properties and time-lapse changes in dynamics revealed a protein-protein interaction in response to ligand stimulation.

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.

Considerations and protocols for the synthesis of custom protein labeling probes.

Chemists and biologists have long recognized small molecule probes as powerful tools for functional genomics and proteomics studies. The possibility of specifically attaching chemical probes to individual proteins with spatial and temporal resolution has greatly improved our ability to visualize and characterize proteins in their native environment. The continued development of novel molecular probes for protein labeling is, therefore, of fundamental importance to gain new insights into biological processes in living cells and organisms. Several excellent approaches for the site-specific labeling of fusion proteins with chemical probes exist. Herein I discuss the design and generation of chemical probes for the SNAP-tag and CLIP-tag systems. The first part of this chapter is dedicated to reviewing the principles of the SNAP-tag technology, followed by a section dedicated to the development of chemical probes for unique applications, such as super-resolution imaging, protein trafficking and recycling, protein-protein interactions, and biomolecular sensing. The last part of the chapter contains experimental protocols and technical notes for the synthesis of selected SNAP-tag substrates and labeling of SNAP-tag fusion proteins in vitro and in living cells.

A Rapid and Fluorogenic TMP-AcBOPDIPY Probe for Covalent Labeling of Proteins in Live Cells

Protein labeling is enormously useful for characterizing protein function in cells and organisms. Chemical tagging methods have emerged as a new generation protein labeling strategy in live cells. Here we have developed a novel and versatile TMP-AcBOPDIPY probe for selective and turn-on labeling of proteins in live cells. A small monomeric tag, E. coli dihydrofolate reductase (eDHFR), was rationally designed to introduce a cysteine in the vicinity of the ligand binding site. Trimethoprim (TMP) that specifically binds to eDHFR was linked to the BOPDIPY fluorophore containing a mildly thiol-reactive acrylamide group. TMP-AcBOPDIPY rapidly labeled engineered eDHFR tags via a reaction termed affinity conjugation (a half-life of ca. 2 min), which is one of the top fast chemical probes for protein labeling. The probe displays 2-fold fluorescence enhancement upon labeling of proteins. We showed that the probe specifically labeled intracellular proteins in live cells without and with washing out the dye. We demonstrated its utility in visualizing intracellular processes by fluorescence-lifetime imaging microscopy (FLIM) measurements.

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.

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Selective Recognition and Detection of Biomacromolecules Utilizing Chemical Property of Amino Acid or Peptide

Recently, the fluorometric detection of biomacromolecules has attracted much attention. In this paper, we report the development of two new techniques utilizing the chemical properties of amino acids or peptides: 1) a fluorescence assay for serine/threonine kinase activity; and 2) "turn-on" fluorescent probes for protein labeling, which could be useful for bioimaging. To develop the novel kinase assay, we utilized the chemical reactivity of phosphorylated serine or threonine. Phosphorylated peptide on resin was successfully labeled fluorescently via base-mediated beta-elimination, followed by Michael addition with novel coumarin derivatives. Protein kinase A and casein kinase I activities were detectable with our method. Also, this method was confirmed to be applicable for kinase inhibitor screening. For the development of the novel protein labeling technique, the selective interaction between "His-tag (His(6))" and "metal ion nitrilotriacetic acid (NTA) complex" was utilized. This interaction is useful for protein purification and immobilization. We designed fluorescent probes composed of a fluorophore and Ni2+ or Co2+-NTA complex. These probes were found to be weakly fluorescent as expected. When His-tag peptide was added, these probes became brightly fluorescent. On the other hand, these probes remained non fluorescent with the addition of angiotensin I (H-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-OH). These probes will be powerful tools for the bioimaging of target proteins.

Reactivity of functional groups on the protein surface: development of epoxide probes for protein labeling.

We present the development of new affinity probes for protein labeling based on an epoxide reactive group. Systematic screening revealed that an epoxide functionality possesses the special combination of stability and reactivity which renders it stable toward proteins in solution but reactive on the protein surface outside the active site (proximity-induced reactivity). Highly efficient and selective labeling of purified HCA II (human carbonic anhydrase II) was achieved. For instance, 2 equiv of epoxide probe 9 was sufficient for nearly quantitative labeling of HCA II (>90% yield, 20 h reaction time). MS analysis of the labeled protein revealed that 1 equiv of the probe was attached and that labeling occurred at a single residue (His 64) outside the active site. Importantly, epoxide probe 9 selectively labeled HCA II both in simple protein mixtures and in cellular extracts. In addition to the chemical insight and its relevance to many epoxide-containing natural products, this study generated a promising lead in the development of new affinity probes for protein labeling.

Organization of membrane proteins in the intact myelin sheath. Pyridoxal phosphate and salicylaldehyde as probes of myelin structure.

Pyridoxal phosphate and salicylaldehyde were used as protein-labeling probes to study the organization of membrane proteins in the intact myelin sheath of the cat dorsal column. Both reagents react with protein amino groups to form Schiff's bases which can be reduced with NaB3H4. The relatively membrane-impermeant pyridoxal phosphate labels all proteins of the intact myelin except basic protein. This major protein of myelin is labeled only after loss of membrane integrity. The relatively membrane-permeant probe, salicylaldehyde, was then used to establish that the basic protein is truly located on the cytoplasmic side of the myelin bilayer, and not merely sequestered within the multiple lamellar structure of the sheath. All proteins in the intact myelin are readily labeled by this reagent, with the label distribution pattern identical to that of disrupted myelin fragments. These data suggest a model for myelin structure in which the basic protein is the only major protein component located exclusively on the cytoplasmic side of the membrane (the major period zone of the sheath), with the other major proteins disposed wholly, or in part, in the extracellular half of the membrane bilayer (the intraperiod zone). All proteins, although asymmetrically disposed with respect to membrane sidedness, appear to be randomly distributed throughout the lamellae which comprise the sheath.