Design of a protein tag and fluorogenic probe with modular structure for live-cell imaging of intracellular proteins.
ABSTRACT: Conditional fluorescence imaging is a powerful technique for precise spatiotemporal analysis of proteins in live cells upon administration of a synthetic probe. To be applicable to various biological phenomena, probes must be membrane-permeable, have a high signal-to-noise ratio, and work quickly. To date, few probes meet all of these requirements. Here, we designed a fluorogenic probe (AcFCANB) that could label intracellular proteins fused to the photoactive yellow protein (PYP) tag in live cells within 30 min and used it to image heterochromatin protein 1 localization in nuclei. AcFCANB is based on a modular platform consisting of fluorophore, ligand and quencher. We accelerated the labeling reaction by strategic mutations of charged residues on the surface of PYP. A simple model based on molecular dynamics simulations quantitatively reproduced the cooperative effect of multiple mutations on labeling rate.
Project description:The ability to specifically attach chemical probes to individual proteins represents a powerful approach to the study and manipulation of protein function in living cells. It provides a simple, robust and versatile approach to the imaging of fusion proteins in a wide range of experimental settings. However, a potential drawback of detection using chemical probes is the fluorescence background from unreacted or nonspecifically bound probes. In this report we present the design and application of novel fluorogenic probes for labeling SNAP-tag fusion proteins in living cells. SNAP-tag is an engineered variant of the human repair protein O(6)-alkylguanine-DNA alkyltransferase (hAGT) that covalently reacts with benzylguanine derivatives. Reporter groups attached to the benzyl moiety become covalently attached to the SNAP tag while the guanine acts as a leaving group. Incorporation of a quencher on the guanine group ensures that the benzylguanine probe becomes highly fluorescent only upon labeling of the SNAP-tag protein. We describe the use of intramolecularly quenched probes for wash-free labeling of cell surface-localized epidermal growth factor receptor (EGFR) fused to SNAP-tag and for direct quantification of SNAP-tagged ?-tubulin in cell lysates. In addition, we have characterized a fast-labeling variant of SNAP-tag, termed SNAP(f), which displays up to a tenfold increase in its reactivity towards benzylguanine substrates. The presented data demonstrate that the combination of SNAP(f) and the fluorogenic substrates greatly reduces the background fluorescence for labeling and imaging applications. This approach enables highly sensitive spatiotemporal investigation of protein dynamics in living cells.
Project description:Developed to complement the use of fluorescent proteins in live cell imaging, chemical tags enjoy the benefit of modular incorporation of organic fluorophores, opening the possibility of high photon output and special photophysical properties. However, the theoretical challenge in using chemical tags as opposed to fluorescent proteins for high-resolution imaging is background noise from unbound and/or nonspecifically bound ligand-fluorophore. We envisioned we could overcome this limit by engineering fluorogenic trimethoprim-based chemical tags (TMP-tags) in which the fluorophore is quenched until binding with E. coli dihydrofolate reductase (eDHFR)-tagged protein displaces the quencher. Thus, we began by building a nonfluorogenic, covalent TMP-tag based on a proximity-induced reaction known to achieve rapid and specific labeling both in vitro and inside of living cells. Here we take the final step and render the covalent TMP-tag fluorogenic. In brief, we designed a trimeric TMP-fluorophore-quencher molecule (TMP-Q-Atto520) with the quencher attached to a leaving group that, upon TMP binding to eDHFR, would be cleaved by a cysteine residue (Cys) installed just outside the binding pocket of eDHFR. We present the in vitro experiments showing that the eDHFR:L28C nucleophile cleaves the TMP-Q-Atto520 rapidly and efficiently, resulting in covalent labeling and remarkable fluorescence enhancement. Most significantly, while only our initial design, TMP-Q-Atto520 achieved the demanding goal of not only labeling highly abundant, localized intracellular proteins but also less abundant, more dynamic cytoplasmic proteins. These results suggest that the fluorogenic TMP-tag can significantly impact high-resolution live cell imaging and further establish the potential of proximity-induced reactivity and organic chemistry more broadly as part of the growing toolbox for synthetic biology and cell engineering.
Project description:Characterization of the chemical environment, movement, trafficking and interactions of proteins in live cells is essential to understanding their functions. Labeling protein with functional molecules is a widely used approach in protein research to elucidate the protein location and functions both in vitro and in live cells or in vivo. A peptide or a protein tag fused to the protein of interest and provides the opportunities for an attachment of small molecule probes or other fluorophore to image the dynamics of protein localization. Here we reviewed the recent development of no-wash small molecular probes for photoactive yellow protein (PYP-tag), by the means of utilizing a quenching mechanism based on the intramolecular interactions, or an environmental-sensitive fluorophore. Several fluorogenic probes have been developed, with fast labeling kinetics and cell permeability. This technology allows quick live-cell imaging of cell-surface and intracellular proteins without a wash-out procedure.
Project description: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'.
Project description:Herein, we present the synthesis and application of a fluorogenic, large Stokes-shift (>100 nm), bioorthogonally conjugatable, membrane-permeable tetrazine probe, which can be excited at common laser line 488 nm and detected at around 600 nm. The applied design enabled improved fluorogenicity in the orange/red emission range, thus efficient suppression of background and autofluorescence upon imaging biological samples. Moreover, unlike our previous advanced probes, it does not require the presence of special target platforms or microenvironments to achieve similar fluorogenicity and can be generally applied, e.g., on translationally bioorthogonalized proteins. Live-cell labeling schemes revealed that the fluorogenic probe is suitable for specific labeling of intracellular proteins, site-specifically modified with a cyclooctynylated, non-canonical amino acid, even under no-wash conditions. Furthermore, the probe was found to be applicable in stimulated emission depletion (STED) super-resolution microscopy imaging using a 660 nm depletion laser. Probably the most salient feature of this new probe is that the large Stokes-shift allows dual-color labeling schemes of cellular structures using distinct excitation and the same detection wavelengths for the combined probes, which circumvents chromatic aberration related problems.
Project description:Genetically encoded fluorescent proteins or small-molecule probes that recognize specific protein binding partners can be used to label proteins to study their localization and function with fluorescence microscopy. However, these approaches are limited in signal-to-background resolution and the ability to temporally control labeling. Herein, we describe a covalent protein labeling technique using a fluorogenic malachite green probe functionalized with a photoreactive cross-linker. This enables a controlled covalent attachment to a genetically encodable fluorogen activating protein (FAP) with low background signal. We demonstrate covalent labeling of a protein in vitro as well as in live mammalian cells. This method is straightforward, displays high labeling specificity, and results in improved signal-to-background ratios in photoaffinity labeling of target proteins. Additionally, this probe provides temporal control over reactivity, enabling future applications in real-time monitoring of cellular events.
Project description:Fluorogenic probes for bioorthogonal labeling chemistry are highly beneficial to reduce background signal in fluorescence microscopy imaging. 1,2,4,5-Tetrazines are known substrates for the bioorthogonal inverse electron demand Diels-Alder reaction (DAinv) and tetrazine substituted fluorophores can exhibit fluorogenic properties. Herein, we report the synthesis of a palette of novel fluorogenic tetrazine dyes derived from widely-used fluorophores that cover the entire emission range from green to far-red. We demonstrate the power of the new fluorogenic probes in fixed and live cell labeling experiments and present the first example of intracellular live cell protein imaging using tetrazine-based probes under no-wash conditions.
Project description:The design of fluorogenic probes for a Halo tag is highly desirable but challenging. Previous work achieved this goal by controlling the chemical switch of spirolactones upon the covalent conjugation between the Halo tag and probes or by incorporating a "channel dye" into the substrate binding tunnel of the Halo tag. In this work, we have developed a novel class of Halo-tag fluorogenic probes that are derived from solvatochromic fluorophores. The optimal probe, harboring a benzothiadiazole scaffold, exhibits a 1000-fold fluorescence enhancement upon reaction with the Halo tag. Structural, computational, and biochemical studies reveal that the benzene ring of a tryptophan residue engages in a cation-? interaction with the dimethylamino electron-donating group of the benzothiadiazole fluorophore in its excited state. We further demonstrate using noncanonical fluorinated tryptophan that the cation-? interaction directly contributes to the fluorogenicity of the benzothiadiazole fluorophore. Mechanistically, this interaction could contribute to the fluorogenicity by promoting the excited-state charge separation and inhibiting the twisting motion of the dimethylamino group, both leading to an enhanced fluorogenicity. Finally, we demonstrate the utility of the probe in no-wash direct imaging of Halo-tagged proteins in live cells. In addition, the fluorogenic nature of the probe enables a gel-free quantification of fusion proteins expressed in mammalian cells, an application that was not possible with previously nonfluorogenic Halo-tag probes. The unique mechanism revealed by this work suggests that incorporation of an excited-state cation-? interaction could be a feasible strategy for enhancing the optical performance of fluorophores and fluorogenic sensors.
Project description:Fluorogenic labeling enables imaging cellular molecules of interest with minimal background. This process is accompanied with the notable increase of the quantum yield of fluorophore, thus minimizing the background signals from unactivated profluorophores. Herein, the development of a highly efficient and bioorthogonal nitroso-based Diels-Alder fluorogenic reaction is presented and its usefulness is validated as effective and controllable in fluorescent probes and live-cell labeling strategies for dynamic cellular imaging. It is demonstrated that nitroso-based cycloaddition is an efficient fluorogenic labeling tool through experiments of further UV-activatable fluorescent labeling on proteins and live cells. The ability of tuning the fluorescence of labeled proteins by UV-irradiation enables selective activation of proteins of interest in a particular cell compartment at a given time point, while leaving the remaining labeled molecules untouched.
Project description:Control and manipulation of bacterial populations requires an understanding of the factors that govern growth, division, and antibiotic action. Fluorescent and chemically reactive small molecule probes of cell envelope components can visualize these processes and advance our knowledge of cell envelope biosynthesis (e.g., peptidoglycan production). Still, fundamental gaps remain in our understanding of the spatial and temporal dynamics of cell envelope assembly. Previously described reporters require steps that limit their use to static imaging. Probes that can be used for real-time imaging would advance our understanding of cell envelope construction. To this end, we synthesized a fluorogenic probe that enables continuous live cell imaging in mycobacteria and related genera. This probe reports on the mycolyltransferases that assemble the mycolic acid membrane. This peptidoglycan-anchored bilayer-like assembly functions to protect these cells from antibiotics and host defenses. Our probe, quencher-trehalose-fluorophore (QTF), is an analog of the natural mycolyltransferase substrate. Mycolyltransferases process QTF by diverting their normal transesterification activity to hydrolysis, a process that unleashes fluorescence. QTF enables high contrast continuous imaging and the visualization of mycolyltransferase activity in cells. QTF revealed that mycolyltransferase activity is augmented before cell division and localized to the septa and cell poles, especially at the old pole. This observed localization suggests that mycolyltransferases are components of extracellular cell envelope assemblies, in analogy to the intracellular divisomes and polar elongation complexes. We anticipate QTF can be exploited to detect and monitor mycobacteria in physiologically relevant environments.