Photoproteins as luminescent labels in binding assays

Certain marine organisms produce calcium-activated photoproteins that allow them to emit light for a variety of purposes, such as defense, feeding, breeding, etc. Even though there are many bioluminescent organisms in nature, only a few photoproteins have been isolated and characterized. The mechanism of emission of light in the blue region is the result of an internal chemical reaction. Because there is no need for excitation through external irradiation for the emission of bioluminescence, the signal produced has virtually no background. This allows for the detection of the proteins at extremely low levels, making these photoproteins attractive labels for analytical applications. In that regard, the use of certain photoproteins, namely, aequorin, obelin, and the green fluorescent protein as labels in the design and development of binding assays for biomolecules has been reviewed. In addition, a related fluorescent photoprotein, the green fluorescent protein (GFP), has been recently employed in bioanalysis. The use of GFP in binding assays is also discussed in this review.     

Fluorescent Protein–photoprotein Fusions and Their Applications in Calcium Imaging

Calcium‐activated photoproteins, such as aequorin, have been used as luminescent Ca²⁺ indicators since 1967. After the cloning of aequorin in 1985, microinjection was substituted by its heterologous expression, which opened the way for a widespread use. Molecular fusion of green fluorescent protein (GFP) to aequorin recapitulated the nonradiative energy transfer process that occurs in the jellyfish Aequorea victoria, from which these two proteins were obtained, resulting in an increase of light emission and a shift to longer wavelength. The abundance and location of the chimera are seen by fluorescence, whereas its luminescence reports Ca²⁺ levels. GFP‐aequorin is broadly used in an increasing number of studies, from organelles and cells to intact organisms. By fusing other fluorescent proteins to aequorin, the available luminescence color palette has been expanded for multiplexing assays and for in vivo measurements. In this report, we will attempt to review the various photoproteins available, their reported fusions with fluorescent proteins and their biological applications to image Ca²⁺ dynamics in organelles, cells, tissue explants and in live organisms.     

GREEN FLUORESCENT PROTEIN

Abstract— Several bioluminescent coelenterates use a secondary fluorescent protein, the green fluorescent protein (GFP), in an energy transfer reaction to produce green light. The most studied of these proteins have been the GFPs from the jellyfish Aequorea victoria and the sea pansy Renilla reniformis. Although the proteins from these organisms are not identical, they are thought to have the same chro-mophore, which is derived from the primary amino acid sequence of GFP. The differences are thought to be due to changes in the protein environment of the chromophore. Recent interest in these molecules has arisen from the cloning of the Aequorea gfp cDNA and the demonstration that its expression in the absence of other Aequorea proteins results in a fluorescent product. This demonstration indicated that GFP could be used as a marker of gene expression and protein localization in living and fixed tissues. Bacterial, plant and animal (including mammalian) cells all express GFP. The heterologous expression of the gfp cDNA has also meant that it could be mutated to produce proteins with different fluorescent properties. Variants with more intense fluorescence or alterations in the excitation and emission spectra have been produced.     

A Far‐Red Fluorescent DNA Binder for Interaction Studies of Live Multidrug‐Resistant Pathogens and Host Cells

Transgene expression of green fluorescent protein (GFP) has facilitated the spatiotemporal investigation of host–pathogen interactions; however, introduction of the GFP gene remains challenging in drug‐resistant bacteria. Herein, we report a novel far‐red fluorescent nucleic acid stain, 6‐TramTO‐3 , which efficiently labels bacteria through a DNA binding mode without affecting growth and viability. Exemplarily, we stained Klebsiella pneumoniae, a major threat to hospitalized patients, and deciphered divergent interaction strategies of antibiotic‐resistant and antibiotic‐sensitive Klebsiella strains with immune cells. 6‐TramTO‐3 constitutes an off‐the‐shelf reagent for real‐time analysis of bacterial infection, including strains for which the use of genetically encoded reporters is not feasible. Eventually, our approach may aid the development of strategies to combat a major worldwide health threat: multidrug‐resistant bacteria.     

Luminescent proteins from Aequorea victoria: applications in drug discovery and in high throughput analysis

Recent progress in generating a vast number of drug targets through genomics and large compound libraries through combinatorial chemistry have stimulated advancements in drug discovery through the development of new high throughput screening (HTS) methods. Automation and HTS techniques are also highly desired in fields such as clinical diagnostics. Luminescence-based assays have emerged as an alternative to radiolabel-based assays in HTS as they approach the sensitivity of radioactive detection along with ease of operation, which makes them amenable to miniaturization. Luminescent proteins provide the advantage of reduced reagent and operating costs because they can be produced in unlimited amounts through the use of genetic engineering tools. In that regard, the use of two naturally occurring and recombinantly produced luminescent proteins from the jellyfish Aequorea victoria, namely, aequorin and the green fluorescent protein (GFP), has attracted attention in a number of analytical applications in diverse research areas. Aequorin is naturally bioluminescent and has therefore, virtually no associated background signal, which allows its detection down to attomole levels. GFP has become the reporter of choice in a variety of applications given that it is an autofluorescent protein that does not require addition of any co-factors for fluorescence emission. Furthermore, the generation of various mutants of GFP with differing luminescent and spectral properties has spurred additional interest in this protein. In this review, we focus on the use of aequorin and GFP in the development of highly sensitive assays that find applications in drug discovery and in high throughput analysis.     

Luminescent proteins from Aequorea victoria: applications in drug discovery and in high throughput analysis

Recent progress in generating a vast number of drug targets through genomics and large compound libraries through combinatorial chemistry have stimulated advancements in drug discovery through the development of new high throughput screening (HTS) methods. Automation and HTS techniques are also highly desired in fields such as clinical diagnostics. Luminescence-based assays have emerged as an alternative to radiolabel-based assays in HTS as they approach the sensitivity of radioactive detection along with ease of operation, which makes them amenable to miniaturization. Luminescent proteins provide the advantage of reduced reagent and operating costs because they can be produced in unlimited amounts through the use of genetic engineering tools. In that regard, the use of two naturally occurring and recombinantly produced luminescent proteins from the jellyfish Aequorea victoria, namely, aequorin and the green fluorescent protein (GFP), has attracted attention in a number of analytical applications in diverse research areas. Aequorin is naturally bioluminescent and has therefore, virtually no associated background signal, which allows its detection down to attomole levels. GFP has become the reporter of choice in a variety of applications given that it is an autofluorescent protein that does not require addition of any co-factors for fluorescence emission. Furthermore, the generation of various mutants of GFP with differing luminescent and spectral properties has spurred additional interest in this protein. In this review, we focus on the use of aequorin and GFP in the development of highly sensitive assays that find applications in drug discovery and in high throughput analysis.     

Synthesis and spectroscopic studies of model red fluorescent protein chromophores

[reaction: see text]. Here we describe the synthesis and spectroscopic characterization of two compounds designed to model the chromophore in DsRed, a red fluorescent protein. Comparison with model green fluorescent protein (GFP) chromophores indicates that the additional conjugation in the DsRed models can account, in part, for the red-shifted absorption and emission properties of DsRed compared to those of GFP. In contrast to the GFP models, the DsRed models are fluorescent with quantum yields of 0.002-0.01 in CHCl3.     

QM/MM Study on the Light Emitters of Aequorin Chemiluminescence,Bioluminescence, and Fluorescence: A General Understanding of the Bioluminescence of Several Marine Organisms

Aequorea victoria is a type of jellyfish that is known by its famous protein, green fluorescent protein (GFP), which has been widely used as a probe in many fields. Aequorea has another important protein, aequorin, which is one of the members of the EF‐hand calcium‐binding protein family. Aequorin has been used for intracellular calcium measurements for three decades, but its bioluminescence mechanism remains largely unknown. One of the important reasons is the lack of clear and reliable knowledge about the light emitters, which are complex. Several neutral and anionic forms exist in chemiexcited, bioluminescent, and fluorescent states and are connected with the H‐bond network of the binding cavity in the protein. We first theoretically investigated aequorin chemiluminescence, bioluminescence, and fluorescence in real proteins by performing hybrid quantum mechanics and molecular mechanics methods combined with a molecular dynamics method. For the first time, this study reported the origin and clear differences in the chemiluminescence, bioluminescence and fluorescence of aequorin, which is important for understanding the bioluminescence not only of jellyfish, but also of many other marine organisms (that have the same coelenterazine caved in different coelenterazine‐type luciferases).     

Nanobody‐Conjugated Nanotubes for Targeted Near‐Infrared In Vivo Imaging and Sensing

Fluorescent nanomaterials such as single‐walled carbon nanotubes (SWCNTs) have many advantages in terms of their photophysics, but it is difficult to target them to specific locations in living systems. In contrast, the green fluorescent protein (GFP) has been genetically fused to proteins in many cells and organisms. Therefore, GFP can be seen not only as a fluorophore but as a universal target/handle. Here, we report the conjugation of GFP‐binding nanobodies to DNA‐wrapped SWCNTs. This approach combines the targeting capabilities of GFP‐binding nanobodies and the nonbleaching near‐infrared fluorescence (850–1700 nm) of SWCNTs. These conjugates allow us to track single Kinesin‐5‐GFP motor proteins in developing embryos of Drosophila melanogaster. Additionally, they are sensitive to the neurotransmitter dopamine and can be used for targeted sensing of dopamine in the nm regime.     

The enhanced green fluorescent protein as a tool for the analysis of protein dynamics and localization: local fluorescence study at the single-molecule level

The green fluorescent protein (GFP) has emerged, in recent years, as a powerful reporter molecule for monitoring gene expression, protein localization and protein-protein interaction. Several mutant variants are now available differing in absorption, emission spectra and quantum yield. Here we present a detailed study of the fluorescence properties of the Phe-64-->Leu, Ser-65-->Thr mutant down to the single molecule level in order to assess its use in quantitative fluorescence microscopy and single-protein trafficking. This enhanced GFP (EGFP) is being used extensively as it offers higher-intensity emission after blue-light excitation with respect to wild-type GFP. By means of fluorescence spectroscopy we demonstrate the absence of the neutral form of the chromophore and the lack of photobleaching recovery after ultraviolet light irradiation. Furthermore, we show that the EGFP spectral properties from isolated to densely packed molecules are highly conserved. From these experiments EGFP emerges as an ideal molecule for quantitative studies of intra and intercellular tagged-protein dynamics and fluorescence-activated cell sorting, but not for monitoring single-protein trafficking over extended periods of time.     

Green fluorescent protein based pH indicators for in vivo use: a review

Green fluorescent protein (GFP) and its variants have been used as fluorescent reporters in a variety of applications for monitoring dynamic processes in cells and organisms, including gene expression, protein localization, and intracellular dynamics. GFP fluorescence is stable, species-independent, and can be monitored noninvasively in living cells by fluorescence microscopy, flow cytometry, or macroscopic imaging techniques. Owing to the presence of a phenol group on the chromophore, most GFP variants display pH-sensitive absorption and fluorescence bands. Such behavior has been exploited to genetically engineer encodable pH indicators for studies of pH regulation within specific intracellular compartments that cannot be probed using conventional pH-sensitive dyes. These pH indicators contributed to shedding light on a number of cell functions for which intracellular pH is an important modulator. In this review we discuss the photophysical properties that make GFPs so special as pH indicators for in vivo use and we describe the probes that are utilized most by the scientific community.     

A FLASH of insight into cellular chemistry: genetically encoded labels for protein visualization in vivo

Genetically encoded fluorescent labels, such as green fluorescent protein, make it possible to visualize a protein's natural distribution and environment in living cells. A new approach to protein labeling in living cells has been devised in which a small, membrane-permeable ligand binds with high affinity and specificity to a short peptide motif that can be incorporated into the protein of interest; the ligand becomes brightly fluorescent after binding to the peptide.     

绿色荧光蛋白

绿色荧光蛋白是46多年前从多管水母体内发现的,它可以在蓝光或紫外光激发下发射绿光.由于它稳定的结构和光物理性质,又易于在细胞内表达,近些年作为标记物已经被广泛地应用于生命科学领域.本文简要介绍了水母发光蛋白与绿色荧光蛋白的关系、绿色荧光蛋白的结构、发色团的形成、发光机制、变异体以及它的特点和应用.     

Real-time measurement of endosomal acidification by a novel genetically encoded biosensor

Genetically encoded fluorescent proteins are optimal reporters when used to monitor cellular processes as they can be targeted to any subcellular region by fusion to a protein of interest. Here, we present the pH-sensitive fluorescent protein E¹GFP which is ideally suited to monitor pH changes in dynamic intracellular structures in real time with high spatio temporal resolution. E¹GFP is a ratiometric pH indicator by emission with a pK close to 6.0. We describe an application of this novel pH reporter in the measurement of pH changes along the endo-lysosomal pathway. By fusing E¹GFP to the HIV-Tat protein which is endowed with cell-penetrating properties, we were able to monitor multi-step endocytosis from the initial cell-surface binding through to the intracellular endocytic network in real time. This represents a framework for the application of E¹GFP to the in situ detection of pH changes involved in dynamic biological phenomena. Figure The green fluorecent protein variant, E1GFP, is a ratiometric pH-indicator by emission with a pK close to 6.0 and is therefore particularly suitable for pH detection below neutrality. Upon excitation of the neutral state of the chromophore (~400-410 nm), E1GFP emission properties are strongly dependent on the environmental pH. We describe an application of this novel pH-reporter in the measurement of pH changes along the endo-lysosomal pathway. By fusing E1GFP to the HIV-Tat protein, which is endowed with cell-penetrating properties, we were able to monitor in real-time multi-step endocytosis from the initial cell-surface binding through to the intracellular endocytic network. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

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.     

Confocal/TEM overlay microscopy: a simple method for correlating confocal and electron microscopy of cells expressing GFP/YFP fusion proteins.

Genetic manipulation allows simultaneous expression of green fluorescent protein (GFP) and its derivatives with a wide variety of cellular proteins in a variety of living systems. Epifluorescent and confocal laser scanning microscopy (confocal) localization of GFP constructs within living tissue and cell cultures has become routine, but correlation of light microscopy and high resolution transmission electron microscopy (TEM) on components within identical cells has been problematic. In this study, we describe an approach that specifically localizes the position of GFP/yellow fluorescent protein (YFP) constructs within the same cultured cell imaged in the confocal and transmission electron microscopes. We present a simplified method for delivering cell cultures expressing fluorescent fusion proteins into LR White embedding media, which allows excellent GFP/YFP detection and also high-resolution imaging in the TEM. Confocal images from 0.5-microm-thick sections are overlaid atop TEM images of the same cells collected from the next serial ultrathin section. The overlay is achieved in Adobe Photoshop by making the confocal image somewhat transparent, then carefully aligning features within the confocal image over the same features visible in the TEM image. The method requires no specialized specimen preparation equipment; specimens are taken from live cultures to embedding within 8 h, and confocal transmission overlay microscopy can be completed within a few hours.     

Quantitative analysis of a ubiquitin‐dependent substrate using capillary electrophoresis with dual laser‐induced fluorescence

Protein degradation by the ubiquitin‐proteasome system (UPS) affects many biological processes. Inhibition of the proteasome has emerged as a potential therapeutic target for cancer treatment. In this study, we developed a method for monitoring the degradation and accumulation of UPS‐dependent substrates in cells using CE with dual LIF. We used a green fluorescent protein (GFP)‐fusion of the ubiquitin substrate ribophorin 1 (GFP‐RPN1) along with red fluorescent protein (RFP) as an internal control to normalize transfection efficiency. Determination of GFP‐RPN1 and RFP in cell lysates were performed in an untreated capillary (75 μm × 50 cm) and 100 mM Tris‐CHES buffer (pH 9.0) containing 10 mM SDS. GFP‐RPN1 and RFP fluorescence were detected at excitation wavelengths of 488 and 635 nm, and emission wavelengths of 520 and 675 nm, respectively, without any interference or crosstalk. The intensity of GFP‐RPN1 fluorescence was normalized to that of RFP. Additionally, the proposed approach was used successfully to detect the degradation of GFP‐RPN1 and evaluate proteasome inhibitors. These results show that the developed method is effective and promising for rapid and quantitative monitoring of UPS‐dependent substrates compared to the current common methods, such as immunoblotting and pulse chase assays.     

Unnatural amino acid mutagenesis of green fluorescent protein

Unnatural amino acid mutagenesis has been used to selectively substitute tyrosine 66 of green fluorescent protein (GFP) with five novel amino acids: p-amino-L-phenylalanine, p-methoxy-L-phenylalanine, p-iodo-L-phenylalanine, p-bromo-L-phenylalanine, and L-3-(2-naphthyl)alanine. The absorbance and emission maxima of the resulting mutant GFPs span the range from 375 to 435 nm and 428 to 498 nm, respectively. The spectral properties of the mutant GFPs, including the absorbance and fluorescence maxima and quantum yields, correlate with the structural and electronic properties of the substituents on the amino acids.