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. Received: 7 November 1999 / Revised: 25 January 2000 / Accepted: 26 January 2000     

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.     

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.     

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.     

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.     

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).     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Optical imaging: bacteria,viruses, and mammalian cells encoding light-emitting proteins reveal the locations of primary tumors and metastases in animals

Early detection of tumors and their metastases is crucial for the prognosis of cancer treatment. Traditionally, tumor detection is achieved by various methods, including magnetic resonance imaging and computerized tomography. With the recent cloning, cellular expression, and real-time imaging of light-emitting proteins, such as Renilla luciferase (Ruc), bacterial luciferase (Lux), firefly luciferase (Luc), green fluorescent protein (GFP), or Ruc-GFP fusion protein, significant efforts have been focused on using these marker proteins for tumor detection. It has also been demonstrated that certain bacteria, viruses, and mammalian cells (BVMC), when administered systemically, are able to gain entry and replicate selectively in tumors. In addition, many tissue/tumor specific promoters have been cloned which allow transgene expression specifically in tumor tissues. Therefore, when light-emitting protein encoded BVMC are injected systemically into rodents, tumor-specific marker gene expression is achieved and is detected in real time based on light emission. Consequently, the locations of primary tumors and previously unknown metastases in animals are revealed in vivo. In the future it will likely be feasible to use engineered light-emitting BVMC as probes for tumor detection and as gene-delivery vehicles in vivo for cancer therapy.     

Red-shifted mutants of green fluorescent protein: reversible photoconversions studied by hole-burning and high-resolution spectroscopy

Mutants of green fluorescent protein (GFP) are usually designed to absorb and emit light as “one color” systems, i.e. with a single, photostable conformation of the chromophore. We have studied two red-shifted GFP-mutants (S65T and EYFP) by means of hole-burning and high-resolution optical spectroscopy at low temperature, and compare the data to those previously reported for RS-GFP. We prove that these GFP-mutants are not “one color” systems because they can be reversibly phototransformed from one conformation into another. The results are rationalized in terms of energy-level schemes that are similar to that previously derived by us for wild-type GFP. In these schemes, each mutant can be interconverted by light among at least three conformations that are associated with the protonation-state of the chromophore. The results have relevance for the study of protein–protein interactions by fluorescence resonance energy transfer (FRET), where GFP-mutants of different colors are used as labels in donor–acceptor pairs. Furthermore, we present a detailed mechanism that explains the “on–off” and “blinking” behavior of single GFP-molecules with the proposed energy-level diagrams.     

ATP detection using a label-free DNA aptamer and a cationic tetrahedralfluorene

A simple and sensitive method for ATP detection using a label-free DNA aptamer as the recognition element and ethidium bromide (EB) as the signal reporter is reported. The ATP-binding aptamer undergoes a conformational switch from the aptamer duplex to the aptamer/target complex upon target binding, which induces the fluorescence change of intercalated EB emission. Good selectivity between ATP and CTP, GTP or UTP has been demonstrated, which is due to the specific recognition between the ATP aptamer and ATP. Using EB alone as a signal reporter, the ATP detection limit was estimated to be approximately 0.2 mM. When a light harvesting cationic tetrahedralfluorene was used as an energy donor to sensitize the intercalated EB emission, a 10-fold increase in detection limit and a 2-fold increase in detection selectivity was demonstrated. The sensitivity and selectivity of the tetrahedralfluorene sensitized assay is comparable to or better than most fluorescent ATP assays with multiple labels.