Intracellular protein labeling with prodrug-like probes using a mutant β-lactamase tag

Intracellular protein labeling with small molecular probes that do not require a washing step for the removal of excess probe is greatly desired for real-time investigation of protein dynamics in living cells. Successful labeling of proteins on the cell membrane has been performed using mutant β-lactamase tag (BL-tag) technology. In the present study, intracellular protein labeling with novel cell membrane permeable probes based on β-lactam prodrugs is described. The prodrug-based probes quickly permeated the plasma membranes of living mammalian cells, and efficiently labeled intracellular proteins at low probe concentrations. Because these cell-permeable probes were activated only inside cells, simultaneous discriminative labeling of intracellular and cell surface BL-tag fusion proteins was attained by using cell-permeable and impermeable probes. Thus, this technology enables adequate discrimination of the location of proteins labeled with the same protein tag, in conjunction with different color probes, by dual-color fluorescence. Moreover, the combination of BL-tag technology and the prodrug-based probes enabled the labeling of target proteins without requiring a washing step, owing to the efficient entry of probes into cells and the fast covalent labeling achieved with BL-tag technology after bioactivation. This prodrug-based probe design strategy for BL-tags provides a simple experimental procedure with application to cellular studies with the additional advantage of reduced stress to living cells.     

Labeling proteins with small molecules by site-specific posttranslational modification

We report here the development of a general strategy for site-specific labeling of proteins with small molecules by posttranslational modification enzyme, phosphopantetheinyl transferase Sfp. The target proteins are expressed as fusions to the peptide carrier protein (PCP) excised from nonribosomal peptide synthetase, and Sfp catalyzes the covalent modification of a specific serine residue on PCP by the small molecule-phosphopantetheinyl conjugate. The labeling reaction proceeds with high specificity and efficiency, targeting PCP fusion proteins in the cell lysate. The PCP tag has been shown to be compatible with various proteins, and Sfp-catalyzed PCP modification, compatible with various small-molecule probes conjugated to coenzyme A, highlighting the potential of the PCP tag for site-specific protein labeling with small molecules.     

Redesign of a Fluorogenic Labeling System To Improve Surface Charge,Brightness, and Binding Kinetics for Imaging the Functional Localization of Bromodomains

Protein labeling with fluorogenic probes is a powerful method for the imaging of cellular proteins. The labeling time and fluorescence contrast of the fluorogenic probes are critical factors for the precise spatiotemporal imaging of protein dynamics in living cells. To address these issues, we took mutational and chemical approaches to increase the labeling kinetics and fluorescence intensity of fluorogenic PYP‐tag probes. Because of charge‐reversal mutations in PYP‐tag and probe redesign, the labeling reaction was accelerated by a factor of 18 in vitro, and intracellular proteins were detected with an incubation period of only 1 min. The brightness of the probe both in vitro and in living cells was enhanced by the mutant tag. Furthermore, we applied this system to the imaging analysis of bromodomains. The labeled mutant tag successfully detected the localization of bromodomains to acetylhistone and the disruption of the bromodomain–acetylhistone interaction by a bromodomain inhibitor.     

Redesign of a Fluorogenic Labeling System To Improve Surface Charge,Brightness, and Binding Kinetics for Imaging the Functional Localization of Bromodomains

Protein labeling with fluorogenic probes is a powerful method for the imaging of cellular proteins. The labeling time and fluorescence contrast of the fluorogenic probes are critical factors for the precise spatiotemporal imaging of protein dynamics in living cells. To address these issues, we took mutational and chemical approaches to increase the labeling kinetics and fluorescence intensity of fluorogenic PYP‐tag probes. Because of charge‐reversal mutations in PYP‐tag and probe redesign, the labeling reaction was accelerated by a factor of 18 in vitro, and intracellular proteins were detected with an incubation period of only 1 min. The brightness of the probe both in vitro and in living cells was enhanced by the mutant tag. Furthermore, we applied this system to the imaging analysis of bromodomains. The labeled mutant tag successfully detected the localization of bromodomains to acetylhistone and the disruption of the bromodomain–acetylhistone interaction by a bromodomain inhibitor.     

Triple-color super-resolution imaging of live cells: resolving submicroscopic receptor organization in the plasma membrane

In living color: efficient intracellular covalent labeling of proteins with a photoswitchable dye using the HaloTag for dSTORM super-resolution imaging in live cells is described. The dynamics of cellular nanostructures at the plasma membrane were monitored with a time resolution of a few seconds. In combination with dual-color FPALM imaging, submicroscopic receptor organization within the context of the membrane skeleton was resolved.     

Monitoring Endocytic Trafficking of Anthrax Lethal Factor by Precise and Quantitative Protein Labeling

Coupling the genetic code expansion technique with bioorthogonal reactions enables precise control over the conjugation site as well as the choice of fluorescent probes during protein labeling. However, the advantages of this strategy over bulky and rigid fluorescent proteins (FPs) remain to be fully explored. Here we applied site‐specific bioorthogonal labeling on anthrax lethal factor (LF) to visualize its membrane translocation inside live cells. In contrast to the previously reported FP tags that significantly perturbed LF’s membrane trafficking, our precisely and quantitatively labeled LF exhibited an endocytic activity comparable to wild‐type LF. This allowed time‐lapse imaging of LF’s natural translocation process from host cell membrane to cytosol, which revealed molecular details of its virulence mechanism. Our strategy is generally applicable for monitoring intracellular protein membrane translocation that is difficult to access using conventional protein labeling methodologies.     

Specific and stable fluorescence labeling of histidine-tagged proteins for dissecting multi-protein complex formation

Labeling of proteins with fluorescent dyes offers powerful means for monitoring protein interactions in vitro and in live cells. Only a few techniques for noncovalent fluorescence labeling with well-defined localization of the attached dye are currently available. Here, we present an efficient method for site-specific and stable noncovalent fluorescence labeling of histidine-tagged proteins. Different fluorophores were conjugated to a chemical recognition unit bearing three NTA moieties (tris-NTA). In contrast to the transient binding of conventional mono-NTA, the multivalent interaction of tris-NTA conjugated fluorophores with oligohistidine-tagged proteins resulted in complex lifetimes of more than an hour. The high selectivity of tris-NTA toward cumulated histidines enabled selective labeling of proteins in cell lysates and on the surface of live cells. Fluorescence labeling by tris-NTA conjugates was applied for the analysis of a ternary protein complex in solution and on surfaces. Formation of the complex and its stoichiometry was studied by analytical size exclusion chromatography and fluorescence quenching. The individual interactions were dissected on solid supports by using simultaneous mass-sensitive and multicolor fluorescence detection. Using these techniques, formation of a 1:1:1 stoichiometry by independent interactions of the receptor subunits with the ligand was shown. The incorporation of transition metal ions into the labeled proteins upon labeling with tris-NTA fluorophore conjugates provided an additional sensitive spectroscopic reporter for detecting and monitoring protein-protein interactions in real time. A broad application of these fluorescence conjugates for protein interaction analysis can be envisaged.     

Anionic Conjugated Polyelectrolytes for FRET-based Imaging of Cellular Membrane Potential

We report a Förster resonance energy transfer (FRET)-based imaging ensemble for the visualization of membrane potential in living cells. A water-soluble poly(fluorene-cophenylene) conjugated polyelectrolyte (FsPFc10) serves as a FRET donor to a voltage-sensitive dye acceptor (FluoVolt^™). We observe FRET between FsPFc10 and FluoVolt^™, where the enhancement in FRET-sensitized emission from FluoVolt^™ is measured at various donor/acceptor ratios. At a donor/acceptor ratio of 1, the excitation of FluoVolt^™ in a FRET configuration results in a three-fold enhancement in its fluorescence emission (compared to when it is excited directly). FsPFc10 efficiently labels the plasma membrane of HEK 293T/17 cells and remains resident with minimal cellular internalization for ~ 1.5 h. The successful plasma membrane-associated colabeling of the cells with the FsPFc10-FluoVolt^™ donor-acceptor pair is confirmed by dual-channel confocal imaging. Importantly, cells labeled with FsPFc10 show excellent cellular viability with no adverse effect on cell membrane depolarization. During depolarization of membrane potential, HEK 293T/17 cells labeled with the donor-acceptor FRET pair exhibit a greater fluorescence response in FluoVolt^™ emission relative to when FluoVolt^™ is used as the sole imaging probe. These results demonstrate the conjugated polyelectrolyte to be a new class of membrane labeling fluorophore for use in voltage sensing schemes.     

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.     

线粒体荧光标记的单颗粒水平高通量评估方法

线粒体是真核细胞能量代谢和信号转导的调控中枢,虽然各种灵敏、特异的线粒体荧光标记技术已经被广泛应用于线粒体研究,但仍缺乏单线粒体水平的荧光染色性能评估方法。基于超高灵敏流式检测技术( High sensitivity flow cytometry, HSFCM)能对单个线粒体进行高灵敏、高通量、多参数定量分析的独特优势,本研究发展了一种单线粒体水平的荧光标记高通量评估方法。将携带靶向线粒体绿色荧光蛋白基因的pAcGFP1-Mito质粒转染至人宫颈癌HeLa细胞中,用G418筛选出稳定转染细胞,分别从瞬时转染和稳定转染的细胞中提取线粒体。此外,从未转染质粒的正常HeLa细胞中提取线粒体,分别进行MitoTracker Green标记以及SYTO 62线粒体DNA染色,应用实验室自行研制的超高灵敏流式检测装置在单线粒体水平对这4种线粒体标记方法的荧光亮度、标记效率和稳定性进行评估。实验结果表明,稳定转染细胞中单个线粒体的绿色荧光蛋白(GFP)荧光亮度为瞬时转染的17.7倍,比MitoTracker Green标记的线粒体亮度高约两个数量级,且标记稳定性好。本研究为线粒体标记方法的选择提供了一种先进的分析方法。     

SNAP-tag fluorogenic probes for wash free protein labeling

In this review, we described the design strategies of SNAP-tag fl uorogenic probes with turn-on fl uorescence responses, which minimized the fl uorescence background and allowed for direct imaging in living cells without wash-out steps. These probes can apply in real-time analysis of protein localization, dynamics, and protein– protein interactions in living cells. Furthermore, the excellent fl uorescent properties made it possible to apply some of the probes in super-resolution fl uorescence imaging.     

Stereochemistry-Dependent Labeling of Organelles with a Near-Infrared-Emissive Phosphorus-Bridged Rhodamine Dye in Live-Cell Imaging

The development of near-infrared (NIR) fluorophores that have both excellent chemical stability and photostability, as well as efficient cell permeability, is highly demanded. In this study, we present phospha-rhodamine (POR) dyes which display significantly improved performance for protein labeling. This is achieved by incorporating a 2-carboxy-3-benzothiophenyl group at the 9-position of the xanthene scaffold. The resulting cis and trans isomers were successfully isolated and structurally characterized using X-ray diffraction. The HaloTag ligand conjugates of the two isomers exhibited different staining abilities in live cells. While the cis isomer showed non-specific accumulation on the organelle membranes, the trans isomer selectively labeled the HaloTag-fused proteins, enabling the long-term imaging of cell division and the 5-color imaging of cell organelles. Molecular dynamics simulations of the HaloTag ligand conjugates within the lipid membrane suggested that the cis isomer is more prone to forming oligomers in the membrane. In contrast, the oligomerization of the trans isomer is effectively suppressed by its interaction with the lipid molecules. By taking advantage of the superior labeling performance of the trans isomer and its NIR-emissive properties, multi-color time-lapse super-resolution 3D imaging, namely super-resolution 5D-imaging, of the interconnected network between the endoplasmic reticulum and microtubules was achieved in living cells.     

Design and synthesis of an enzyme activity-based labeling molecule with fluorescence spectral change

Methods of covalent labeling of a specific tag protein with small-molecular dyes play an important role in studying dynamic behaviors of proteins in living cells. On the basis of quinone methide chemistry, we designed and synthesized a beta-galactosidase labeling probe, CMFbeta-gal, which shows a fluorescence wavelength change accompanying the labeling reaction, owing to fluorescence resonance energy transfer (FRET). Since the FRET efficiency changes accompanying the labeling reaction, fluorescence of labeled protein can be observed separately from that of the unreacted probe, so immediate detection of the target protein is possible. This is the first report of a protein labeling probe which features a change of fluorescence wavelength upon reaction, allowing the labeled protein to be detected even in the presence of unreacted probe.     

Fluorescent lipids: functional parts of fusogenic liposomes and tools for cell membrane labeling and visualization

In this paper a rapid and highly efficient method for controlled incorporation of fluorescent lipids into living mammalian cells is introduced. Here, the fluorescent molecules have two consecutive functions: First, they trigger rapid membrane fusion between cellular plasma membranes and the lipid bilayers of their carrier particles, so called fusogenic liposomes, and second, after insertion into cellular membranes these molecules enable fluorescence imaging of cell membranes and membrane traffic processes. We tested the fluorescent derivatives of the following essential membrane lipids for membrane fusion: Ceramide, sphingomyelin, phosphocholine, phosphatidylinositol-bisphosphate, ganglioside, cholesterol, and cholesteryl ester. Our results show that all probed lipids could more efficiently be incorporated into the plasma membrane of living cells than by using other methods. Moreover, labeling occurred in a gentle manner under classical cell culture conditions reducing cellular stress responses. Staining procedures were monitored by fluorescence microscopy and it was observed that sphingolipids and cholesterol containing free hydroxyl groups exhibit a decreased distribution velocity as well as a longer persistence in the plasma membrane compared to lipids without hydroxyl groups like phospholipids or other artificial lipid analogs. After membrane staining, the fluorescent molecules were sorted into membranes of cell organelles according to their chemical properties and biological functions without any influence of the delivery system.     

Use of a solid-state nanopore for profiling the transferrin receptor protein and distinguishing between transferrin receptor and its ligand protein

A nanopore device is capable of providing single-molecule level information of an analyte as they translocate through the sensing aperture—a nanometer-sized through-hole—under the influence of an applied electric field. In this study, a silicon nitride (Si_xN_y)-based nanopore was used to characterize the human serum transferrin receptor protein (TfR) under various applied voltages. The presence of dimeric forms of TfR was found to decrease exponentially as the applied electric field increased. Further analysis of monomeric TfR also revealed that its unfolding behaviors were positively dependent on the applied voltage. Furthermore, a comparison between the data of monomeric TfR and its ligand protein, human serum transferrin (hSTf), showed that these two protein populations, despite their nearly identical molecular weights, could be distinguished from each other by means of a solid-state nanopore (SSN). Lastly, the excluded volumes of TfR were experimentally determined at each voltage and were found to be within error of their theoretical values. The results herein demonstrate the successful application of an SSN for accurately classifying monomeric and dimeric molecules while the two populations coexist in a heterogeneous mixture.     

Live cell labeling of native intracellular bacterial receptors using aniline-catalyzed oxime ligation

Live cell fluorescent labeling of proteins has become a seminal tool in biology and has led to hallmark discoveries in diverse research areas such as protein trafficking, cell-to-cell interactions, and intracellular network dynamics. One of the main challenges, however, remains the ability to label intracellular proteins using fluorescent ligands with high specificity, all the while retaining viability of the targeted cells. Here, we present the first example of live cell labeling and imaging of an intracellular bacterial receptor involved in cell-to-cell communication (i.e., quorum sensing), using a novel two-step approach involving covalent attachment of a reactive mimic of the primary endogenous Pseudomonas aeruginosa quorum-sensing signal to its receptor, LasR, followed by aniline-catalyzed oxime formation between the modified receptor and a fluorescent BODIPY derivative. Our results indicate that LasR is not distributed evenly throughout the cytoplasmic membrane but is instead concentrated at the poles of the P. aeruginosa cell.     

Phagemid encoded small molecules for high throughput screening of chemical libraries

A new strategy for monovalently displaying small molecules on phage surfaces was developed and applied to high throughput screening for molecules with high binding affinity to the target protein. Peptidyl carrier protein (PCP) excised from nonribosomal peptide synthetase was monovalently displayed on the surface of M13 phage as pIII fusion proteins. Small molecules of diverse structures were conjugated to coenzyme A (CoA) and then covalently attached to the phage displayed PCP by Sfp phosphopantetheinyl transferase. Because Sfp is broadly promiscuous for the transfer of small molecule linked phosphopantetheinyl moieties to apo PCP domains, this approach will enable displaying libraries of small molecules on phage surfaces. Unique 20-base-pair (bp) DNA sequences were also incorporated into the phagemid DNA so that each compound displayed on the phage surface was encoded by a DNA bar code encapsulated inside the phage coat protein. Single round selection of phage displayed small molecules achieved more than 2000-fold enrichment of small molecules with nM binding affinity to the target protein. The selection process is further accelerated by the use of DNA decoding arrays for identifying the selected small molecules.     

Peptide‐Templated Acyl Transfer: A Chemical Method for the Labeling of Membrane Proteins on Live Cells

The development of a method is described for the chemical labeling of proteins which occurs with high target specificity, proceeds within seconds to minutes, and offers a free choice of the reporter group. The method relies upon the use of peptide templates, which align a thioester and an N‐terminal cysteinyl residue such that an acyl transfer reaction is facilitated at nanomolar concentrations. The protein of interest is N‐terminally tagged with a 22 aa long Cys‐E3 peptide (acceptor), which is capable of forming a coiled‐coil with a reporter‐armed K3 peptide (donor). This triggers the transfer of the reporter to the acceptor on the target protein. Because ligation of the two interacting peptides is avoided, the mass increase at the protein of interest is minimal. The method is exemplified by the rapid fluorescent labeling and fluorescence microscopic imaging of the human Y₂ receptor on living cells.     

基于迈克尔加成反应的沙门氏菌纳米荧光标记研究

本文通过迈克尔加成反应的荧光标记法,成功构建了纳米荧光探针标记的沙门氏菌(YB1-INPs)。采用三(2-羧乙基)膦将沙门氏菌YB1外膜蛋白表面上的二硫键温和还原为巯基后,与吲哚菁绿(ICG)@纳米探针(INPs)的马来酰亚胺基团(Mal)交联形成YB1-INPs。通过扫描电镜和共聚焦荧光成像对YB1-INPs的形貌、光学性质进行表征。通过活死细菌染色和LB琼脂平板实验对YB1-INPs的活性进行考察。结果表明,INPs成功标记到沙门氏菌YB1表面后对YB1的形态、光学性质、活性及生长均没有产生影响,并且能够用于沙门氏菌YB1体内靶向的荧光分布成像。本研究提供的这种简单、安全、高效的荧光标记方法能够将纳米材料标记到生物载体上,可以应用于生物载体的药物输送和体内监测。     

Real-time measurements of protein dynamics using fluorescence activation-coupled protein labeling method

We present a fluorescence activation-coupled protein labeling (FAPL) method, which employs small-molecular probes that exhibit almost no basal fluorescence but acquire strong fluorescence upon covalent binding to tag-proteins. This method enables real-time imaging of protein labeling without any washout process and is uniquely suitable for real-time imaging of protein dynamics on the cell surface. We applied this method to address the spatiotemporal dynamics of the EGF receptor during cell migration.