Developing Bright Green Fluorescent Protein (GFP)-like Fluorogens for Live-Cell Imaging with Nonpolar Protein−Chromophore Interactions

Fluorescence-activating proteins (FAPs) that bind a chromophore and activate its fluorescence have gained popularity in bioimaging. The fluorescence-activating and absorption-shifting tag (FAST) is a light-weight FAP that enables fast reversible fluorogen binding, thus advancing multiplex and super-resolution imaging. However, the rational design of FAST-specific fluorogens with large fluorescence enhancement (FE) remains challenging. Herein, a new fluorogen directly engineered from green fluorescent protein (GFP) chromophore by a unique double-donor-one-acceptor strategy, which exhibits an over 550-fold FE upon FAST binding and a high extinction coefficient of approximately 100,000 M⁻¹ cm⁻¹, is reported. Correlation analysis of the excited state nonradiative decay rates and environmental factors reveal that the large FE is caused by nonpolar protein−fluorogen interactions. Our deep insights into structure-function relationships could guide the rational design of bright fluorogens for live-cell imaging with extended spectral properties such as redder emissions.     

NanoFAST: structure-based design of a small fluorogen-activating protein with only 98 amino acids

One of the essential characteristics of any tag used in bioscience and medical applications is its size. The larger the label, the more it may affect the studied object, and the more it may distort its behavior. In this paper, using NMR spectroscopy and X-ray crystallography, we have studied the structure of fluorogen-activating protein FAST both in the apo form and in complex with the fluorogen. We showed that significant change in the protein occurs upon interaction with the ligand. While the protein is completely ordered in the complex, its apo form is characterized by higher mobility and disordering of its N-terminus. We used structural information to design the shortened FAST (which we named nanoFAST) by truncating 26 N-terminal residues. Thus, we created the shortest genetically encoded tag among all known fluorescent and fluorogen-activating proteins, which is composed of only 98 amino acids.

We solved the structure of a fluorogen-activating protein FAST and synthesized the library of potential fluorogens. Using these data, we designed the shortest genetically encoded fluorescent tag among all known.     

Selective two-step labeling of proteins with an off/on fluorescent probe

We present a novel design strategy for off/on fluorescent probes suitable for selective two-step labeling of proteins. To validate this strategy, we designed and synthesized an off/on fluorescent probe, 1-Ni(2+), which targets a cysteine-modified hexahistidine (His) tag. The probe consists of dichlorofluorescein conjugated with nitrilotriacetic acid (NTA)-Ni(2+) as the His-tag recognition site and a 2,4-dinitrophenyl ether moiety, which quenches the probe's fluorescence by photoinduced electron transfer (PeT) from the excited fluorophore to the 2,4-dinitrophenyl ether (donor-excited PeT; d-PeT) and also has reactivity with cysteine. His-tag recognition by the NTA-Ni(2+) moiety is followed by removal of the 2,4-dinitrophenyl ether quencher by proximity-enhanced reaction with the cysteine residue of the modified tag; this results in a marked fluorescence increase. Addition of His-tag peptide bearing a cysteine residue to aqueous probe solution resulted in about 20-fold fluorescence increment within 10 min, which is the largest fluorescence enhancement so far obtained with a visible light-excitable fluorescent probe for a His-based peptide tag. Further, we successfully visualized CysHis(6)-peptide tethered to microbeads without any washing step. The probe also showed a large fluorescence increment in the presence of His(6)Cys-tagged enhanced blue fluorescent protein (EBFP), but not His(6)-tagged EBFP. We consider this system is superior to large fluorescence tags (e.g., green fluorescent protein: 27 kDa), which can perturb protein folding, trafficking and function, and also to existing small tags, which generally show little fluorescence increase upon target recognition and therefore require a washout step. This strategy should also be applicable to other tags.     

一种具有双重功能的可视化pH荧光探针的合成及光谱研究

以二烯单酮结构为荧光团,酚羟基为脱质子基团,合成了一种具有双重功能的可视化pH荧光分子探针.pH滴定实验表明,探针的紫外吸收和荧光光谱均对溶液的pH值有很强的依赖性,当体系溶液由酸性变为碱性时,探针的紫外吸收光谱发生明显的红移,并伴有溶液颜色的显著变化;荧光光谱强度在酸性条件下随pH值的变化不大,而在碱性条件下随pH值...     

Coumarin‐Based Fluorogenic Probes for No‐Wash Protein Labeling

A fluorescent protein‐labeling strategy was developed in which a protein of interest (POI) is genetically tagged with a short peptide sequence presenting two Cys residues that can selectively react with synthetic fluorogenic reagents. These fluorogens comprise a fluorophore and two maleimide groups that quench fluorescence until they both undergo thiol addition during the labeling reaction. Novel fluorogens were prepared and kinetically characterized to demonstrate the importance of a methoxy substituent on the maleimide in suppressing reactivity with glutathione, an intracellular thiol, while maintaining reactivity with the dithiol tag. This system allows the rapid and specific labeling of intracellular POIs.     

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.     

Anthozoa red fluorescent protein in biosensing

The identification and cloning of a red fluorescent protein (DsRed) obtained from Anthozoa corals has provided an alternative to commonly used green fluorescent proteins (GFPs) in bioanalytical and biomedical research. DsRed in tandem with GFPs has enhanced the feasibility of multicolor labeling studies. Properties of DsRed, for example high photostability, red-shifted fluorescence emission, and stability to pH changes have proven valuable in its use as a fluorescent tag in cell-biology applications. DsRed has some limitations, however. Its slow folding and tendency to form tetramers have been a hurdle. Several different mutational studies have been performed on DsRed to overcome these problems. In this paper, applications of DsRed in biosensing, specifically in FRET/BRET assays, whole-cell assays, and in biosensors, is discussed. In the future, construction of DsRed mutants with unique characteristics will further expand its applications in bioanalysis.     

Single‐Molecule Localization Microscopy using mCherry

We demonstrate the potential of the commonly used red fluorescent protein mCherry for single‐molecule super‐resolution imaging. mCherry can be driven into a light‐induced dark state in the presence of a thiol from which it can recover spontaneously or by irradiation with near UV light. We show imaging of subcellular protein structures such as microtubules and the nuclear pore complex with a resolution below 40 nm. We were able to image the C‐terminus of the nuclear pore protein POM121, which is on the inside of the pore and not readily accessible for external labeling. The photon yield for mCherry is comparable to that of the latest optical highlighter fluorescent proteins. Our findings show that the widely used mCherry red fluorescent protein and the vast number of existing mCherry fusion proteins are readily amenable to super‐resolution imaging. This obviates the need for generating novel protein fusions that may compromise function or the need for external fluorescent labeling.     

利用聚合物自组装实现绿色荧光蛋白生色团的荧光增强

根据绿色荧光蛋白的发光原理,采用聚乙二醇与聚甲基丙烯酸甲酯的两亲性两嵌段聚合物通过自组装包覆生色团的方式,模拟了绿色荧光蛋白发光,考察了组装行为对光学性能的影响,并将其用于细胞成像.通过核磁共振、高分辨质谱、傅里叶变换红外光谱、凝胶渗透色谱、紫外-可见吸收光谱及荧光光谱等表征了生色团分子和聚合物的结构及性能.生色团紫外最大吸收在371 nm,荧光最大发射峰在428 nm.聚合物和生色团进行组装后,其紫外吸收消失,而最大荧光发射峰强度大大增强,且发生了约70 nm的红移,这是因为组装使得生色团的自由旋转受到了限制,且生色团共平面性增加.动态光散射(DLS)和透射电镜(TEM)证明了纳米粒子的结构和尺寸.由于尺寸适合且具有较好的荧光性能,纳米粒子成功应用于细胞成像.这种绿色荧光蛋白生色团的简单自组装方式在生物成像领域具有良好应用前景.     

A Pyrene-based Probe for Antimony with Special Excimer Fluorescence

Py-IPH, a pyrene-based fluorogen with good thermal and mechanical stability was simply prepared by the Schiff base reaction and applied in the detection of antimony. Py-IPH presented special excimer fluorescence in the mixture of THF and water with 80 % of water, and exhibited perfect fluorescent response to antimony. The mechanism was demonstrated to be the increasing speed of the formation of pyrene excimers in the system owing to the coordination between antimony and IPH group in Py-IPH. Moreover, based on the special interaction between Py-IPH and antimony, it could distinguish antimony with other cations well with high specificity and selectivity.     

Fluorogenic Protein Probes with Red and Near-Infrared Emission for Genetically Targeted Imaging**

Fluorogenic probes are important tools to image proteins with high contrast and no wash protocols. In this work, we rationally designed and synthesized a small set of four protein fluorogens with red or near-infrared emission. The fluorophores were characterized in the presence of albumin as a model protein environment and exhibited good fluorogenicity and brightness (fluorescence quantum yield up to 36 %). Once conjugated to a haloalkane ligand, the probes reacted with the protein self-labeling tag HaloTag with a high fluorescence enhancement (up to 156-fold). The spectroscopic properties of the fluorogens and their reaction with HaloTag were investigated experimentally in vitro and with the help of molecular dynamics. The two most promising probes, one in the red and one in the near-infrared range, were finally applied to image the nucleus or actin in live-cell and in wash-free conditions using fluorogenic and chemogenetic targeting of HaloTag fusion proteins.     

Sensing Protein Surfaces with Targeted Fluorescent Receptors

A methodology for creating fluorescent molecular sensors that respond to changes that occur on the surfaces of specific proteins is presented. This approach, which relies on binding cooperatively between a specific His‐tag binder and a nonspecific protein‐surface receptor, enabled the development of a sensor that can track changes on the surface of a His‐tag‐labeled calmodulin (His‐CaM) upon interacting with metal ions, small molecules, and protein binding partners. The way this approach was used to detect dephosphorylation of an unlabeled calmodulin‐dependent protein kinase II (CaMKII), and the binding of Bax BH3 to His‐tagged B‐cell lymphoma 2 (Bcl‐2) protein is also presented.     

Red‐Shifted Fluorescent Aminated Derivatives of a Conformationally Locked GFP Chromophore

A novel class of fluorescent dyes based on conformationally locked GFP chromophore is reported. These dyes are characterized by red‐shifted spectra, high fluorescence quantum yields and pH‐independence in physiological pH range. The intra‐ and intermolecular mechanisms of radiationless deactivation of ABDI‐BF2 fluorophore by selective structural locking of various conformational degrees of freedom were studied. A unique combination of solvatochromic and lipophilic properties together with “infinite” photostability (due to a dynamic exchange between free and bound dye) makes some of the novel dyes promising bioinspired tools for labeling cellular membranes, lipid drops and other organelles.     

Quantification of Photosensitized Singlet Oxygen Production by a Fluorescent Protein

Fluorescent proteins are increasingly becoming actuators in a range of cell biology techniques. One of those techniques is chromophore‐assisted laser inactivation (CALI), which is employed to specifically inactivate the function of target proteins or organelles by producing photochemical damage. CALI is achieved by the irradiation of dyes that are able to produce reactive oxygen species (ROS). The combination of CALI and the labelling specificity that fluorescent proteins provide is useful to avoid uncontrolled photodamage, although the inactivation mechanisms by ROS are dependent on the fluorescent protein and are not fully understood. Herein, we present a quantitative study of the ability of the red fluorescent protein TagRFP to produce ROS, in particular singlet oxygen (¹O₂). TagRFP is able to photosensitize ¹O₂ with an estimated quantum yield of 0.004. This is the first estimation of a quantum yield of ¹O₂ production value for a GFP‐like protein. We also find that TagRFP has a short triplet lifetime compared to EGFP, which reflects relatively high oxygen accessibility to the chromophore. The insight into the structural and photophysical properties of TagRFP has implications in improving fluorescent proteins for fluorescence microscopy and CALI.     

Mechanically Modulating the Photophysical Properties of Fluorescent Protein Biocomposites for Ratio‐ and Intensiometric Sensors

Mechanically sensitive biocomposites comprised of fluorescent proteins report stress through distinct pathways. Whereas a composite containing an enhanced yellow fluorescent protein (eYFP) exhibited hypsochromic shifts in its fluorescence emission maxima following compression, a composite containing a modified green fluorescent protein (GFPuv) exhibited fluorescence quenching under the action of mechanical force. These ratio‐ and intensiometric sensors demonstrate that insights garnered from disparate fields (that is, polymer mechanochemistry and biophysics) can be harnessed to guide the rational design of new classes of biomechanophore‐containing materials.     

Synthesis and Characterization of Far‐Red/NIR‐Fluorescent BODIPY Dyes,Solid‐State Fluorescence,and Application as Fluorescent Tags Attached to Carbon Nano‐onions

A series of π‐extended distyryl‐substituted boron dipyrromethene (BODIPY) derivatives with intense far‐red/near‐infrared (NIR) fluorescence was synthesized and characterized, with a view to enhance the dye’s performance for fluorescence labeling. An enhanced brightness was achieved by the introduction of two methyl substituents in the meso positions on the phenyl group of the BODIPY molecule; these substituents resulted in increased structural rigidity. Solid‐state fluorescence was observed for one of the distyryl‐substituted BODIPY derivatives. The introduction of a terminal bromo substituent allows for the subsequent immobilization of the BODIPY fluorophore on the surface of carbon nano‐onions (CNOs), which leads to potential imaging agents for biological and biomedical applications. The far‐red/NIR‐fluorescent CNO nanoparticles were characterized by absorption, fluorescence, and Raman spectroscopies, as well as by thermogravimetric analysis, dynamic light scattering, high‐resolution transmission electron microscopy, and confocal microscopy.     

Refolding and purification of histidine-tagged protein by artificial chaperone-assisted metal affinity chromatography

This article has proposed an artificial chaperone-assisted immobilized metal affinity chromatography (AC-IMAC) for on-column refolding and purification of histidine-tagged proteins. Hexahistidine-tagged enhanced green fluorescent protein (EGFP) was overexpressed in Escherichia coli, and refolded and purified from urea-solubilized inclusion bodies by the strategy. The artificial chaperone system was composed of cetyltrimethylammonium bromide (CTAB) and β-cyclodextrin (β-CD). In the refolding process, denatured protein was mixed with CTAB to form a protein–CTAB complex. The mixture was then loaded to IMAC column and the complex was bound via metal chelating to the histidine tag. This was followed by washing with a refolding buffer containing β-CD that removed CTAB from the bound protein and initiated on-column refolding. The effect of the washing time (i.e., on-column refolding time) on mass and fluorescence recoveries was examined. Extensive studies by comparison with other related refolding techniques have proved the advantages of AC-IMAC. In the on-column refolding, the artificial chaperone system suppressed protein interactions and facilitated protein folding to its native structure. So, the on-column refolding by AC-IMAC led to 99% pure EGFP with a fluorescence recovery of 80%. By comparison at a similar final EGFP concentration (0.6–0.8 mg/mL), this fluorescence recovery value was not only much higher than direct dilution (14%) and AC-assisted refolding (26%) in bulk solutions, but also superior to its partner, IMAC (60%). The operating conditions would be further optimized to improve the refolding efficiency.     

Metal Affinity-Based Purification of a Red Fluorescent Protein

A red fluorescent protein, DsRed, which emits fluorescence in the red region of the spectrum has become a popular alternative to green fluorescent protein as a label in biochemical and bioanalytical applications. In this study, we have developed a simple purification method for DsRed variants utilizing their inherent copper binding property. A purification procedure was developed and optimized using immobilized copper ions yielding a single strong band corresponding to purified DsRed proteins on the SDS-PAGE gel. A purification efficiency of higher than 95% was achieved. A spectral analysis and copper binding study was performed to verify activity of the purified proteins. The development of this method allows DsRed to play a dual role as a fluorescent reporter protein and as a purification affinity tag for a target protein. This simpler approach of purification should expand the utility of DsRed.     

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.     

A novel design method of ratiometric fluorescent probes based on fluorescence resonance energy transfer switching by spectral overlap integral

A ratiometric measurement, namely, simultaneous recording of the fluorescence intensities at two wavelengths and calculation of their ratio, allows greater precision than measurements at a single wavelength, and is suitable for cellular imaging studies. Here we describe a novel method of designing probes for ratiometric measurement of hydrolytic enzyme activity based on switching of fluorescence resonance energy transfer (FRET). This method employs fluorescent probes with a 3'-O,6'-O-protected fluorescein acceptor linked to a coumarin donor through a linker moiety. As there is no spectral overlap integral between the coumarin emission and fluorescein absorption, the fluorescein moiety cannot accept the excitation energy of the donor moiety and the donor fluorescence can be observed. After cleavage of the protective groups by hydrolytic enzymes, the fluorescein moiety shows a strong absorption in the coumarin emission region, and then acceptor fluorescence due to FRET is observed. Based on this mechanism, we have developed novel ratiometric fluorescent probes (1-3) for protein tyrosine phosphatase (PTP) activity. They exhibit a large shift in their emission wavelength after reaction with PTPs. The fluorescence quenching problem that usually occurs with FRET probes is overcome by using the coumarin-cyclohexane-fluorescein FRET cassette moiety, in which close contact of the two dyes is hindered. After study of their chemical and kinetic properties, we have concluded that compounds 1 and 2 bearing a rigid cyclohexane linker are practically useful for the ratiometric measurement of PTPs activity. The design concept described in this paper, using FRET switching by spectral overlap integral and a rigid link that prevents close contact of the two dyes, should also be applicable to other hydrolytic enzymes by introducing other appropriate enzyme-cleavable groups into the fluorescein acceptor.