绿色荧光蛋白——照亮生命科学的一盏明灯

绿色荧光蛋白的发现及应用具有划时代的重要意义,它不仅为当代生物学研究提供了极为实用的基本研究手段,并且在此基础上改造发展和发现了一系列荧光蛋白,拓展了应用范围。这使得对微观生物学的研究也可以进入一个时空结合,研究鲜活动态过程的新时代。本文主要回顾总结了绿色荧光蛋白的发现、优化改造及其应用。     

Green fluorescent protein: a perspective

A brief personal perspective is provided for green fluorescent protein (GFP), covering the period 1994-2011. The topics discussed are primarily those in which my research group has made a contribution and include structure and function of the GFP polypeptide, the mechanism of fluorescence emission, excited state protein transfer, the design of ratiometric fluorescent protein biosensors and an overview of the fluorescent proteins derived from coral reef animals. Structure-function relationships in photoswitchable fluorescent proteins and nonfluorescent chromoproteins are also briefly covered.     

Identification of GFP-like Proteins in Nonbioluminescent,Azooxanthellate Anthozoa Opens New Perspectives for Bioprospecting

We screened nonbioluminescent, azooxanthellate cnidaria as potential sources for advanced marker proteins and succeeded in cloning a tetrameric green fluorescent protein (GFP) from the tentacles of Cerianthus membranaceus. The fluorescence of this protein (cmFP512) is characterized by excitation maximum at 503 nm, emission maximum at 512 nm, extinction coefficient of 58,800 M^–1 cm^–1, quantum yield of 0.66, and fluorescence lifetime of 2.4 ns. The chromophore is formed from the tripeptide Gln-Tyr-Gly. The amino acid sequence of this protein shares 17.8% identical residues with GFP from Aequorea victoria. Weak interactions between the subunits of the tetramer make cmFP512 a promising lead structure for the generation of monomeric variants of fluorescent proteins. Both red fluorescent proteins and nonfluorescent proteins of the GFP family were also purified from tissue homogenates of Adamsia palliata and Calliactis parasitica. The results presented here indicate that a photoprotective function of GFP-like proteins is unlikely in the examined anthozoa species.     

Simultaneous detection of DsRed2-tagged and EGFP-tagged human beta-interferons in the same single cells

The red fluorescent protein DsRed2 is a useful fusion tag for various proteins, together with the enhanced green fluorescent protein (EGFP). These chromoproteins have spectral properties that allow simultaneous distinctive detection of tagged proteins in the same single cells by dual color imaging. We used them for tagging a secretory protein, human interferon-beta (IFN-beta). Expression plasmids for human IFN-beta tagged with DsRed2 or with EGFP at the carboxyl terminal were constructed and their coexpression was examined in Mardin-Darby canine kidney epithelial cells. Although maturation of DsRed2 for coloration was slow and the color intensity was weak compared with EGFP, low temperature treatment (20 degrees C) allowed DsRed2-tagged human IFN-beta to be detected in the cells using color imaging. Consequently, the two chimeric proteins were shown to be colocalized in the same single cells by dual color confocal microscopy. This approach will be useful for investigating subcellular localization of not only cell resident proteins but also secretory proteins.     

Diversity and evolution of coral fluorescent proteins

GFP-like fluorescent proteins (FPs) are the key color determinants in reef-building corals (class Anthozoa, order Scleractinia) and are of considerable interest as potential genetically encoded fluorescent labels. Here we report 40 additional members of the GFP family from corals. There are three major paralogous lineages of coral FPs. One of them is retained in all sampled coral families and is responsible for the non-fluorescent purple-blue color, while each of the other two evolved a full complement of typical coral fluorescent colors (cyan, green, and red) and underwent sorting between coral groups. Among the newly cloned proteins are a "chromo-red" color type from Echinopora forskaliana (family Faviidae) and pink chromoprotein from Stylophora pistillata (Pocilloporidae), both evolving independently from the rest of coral chromoproteins. There are several cyan FPs that possess a novel kind of excitation spectrum indicating a neutral chromophore ground state, for which the residue E167 is responsible (numeration according to GFP from A. victoria). The chromoprotein from Acropora millepora is an unusual blue instead of purple, which is due to two mutations: S64C and S183T. We applied a novel probabilistic sampling approach to recreate the common ancestor of all coral FPs as well as the more derived common ancestor of three main fluorescent colors of the Faviina suborder. Both proteins were green such as found elsewhere outside class Anthozoa. Interestingly, a substantial fraction of the all-coral ancestral protein had a chromohore apparently locked in a non-fluorescent neutral state, which may reflect the transitional stage that enabled rapid color diversification early in the history of coral FPs. Our results highlight the extent of convergent or parallel evolution of the color diversity in corals, provide the foundation for experimental studies of evolutionary processes that led to color diversification, and enable a comparative analysis of structural determinants of different colors.     

Natural animal coloration can Be determined by a nonfluorescent green fluorescent protein homolog

It is generally accepted that the colors displayed by living organisms are determined by low molecular weight pigments or chromoproteins that require a prosthetic group. The exception to this rule is green fluorescent protein (GFP) from Aequorea victoria that forms a fluorophore by self-catalyzed protein backbone modification. Here we found a naturally nonfluorescent homolog of GFP to determine strong purple coloration of tentacles in the sea anemone Anemonia sulcata. Under certain conditions, this novel chromoprotein produces a trace amount of red fluorescence (emission lambda(max) = 595 nm). The fluorescence demonstrates unique behavior: its intensity increases in the presence of green light but is inhibited by blue light. The quantum yield of fluorescence can be enhanced dramatically by single amino acid replacement, which probably restores the ancestral fluorescent state of the protein. Other fluorescent variants of the novel protein have emission peaks that are red-shifted up to 610 nm. They demonstrate that long wavelength fluorescence is attainable in GFP-like fluorescent proteins.     

Novel fluorescent protein from Hydnophora rigida possess cyano emission

Currently, a broad diversity of fluorescent proteins among marine organisms range from cyano-red emissions. Fluorescent proteins differ in their DNA sequences from green fluorescent protein (GFP). We identified cDNA encoding the gene of a new protein from the reef coral Hydnophora rigida of the Merulinidae family. Both the spectral properties and putative primary sequence of the protein has been determined. The cloned cDNA encode peptide we call HriCFP is comprised of 134 amino acids. It has characteristics of a cyano fluorescent protein (HriCFP) and its sequence is markedly different from known GFP from the hydroid jellyfish Aequorea victoria. HriCFP was cloned, expressed, purified and exist as monomer. The peptide mass finger print on the purified protein confirmed identity of HriCFP.     

珊瑚和海葵来源红荧光蛋白的研究和应用

绿色荧光蛋白作为标记蛋白和报告蛋白在生物学研究中应用越来越广。但在荧光共振能量转移(fluorescenceresonanceenergytransfer,FRET)等技术中存在一些缺陷,需要更大波长范围的荧光蛋白。最近研究发现了多种来源于珊瑚和海葵的红荧光蛋白,这些长波长的荧光蛋白对绿色荧光蛋白是一种很好的代替和补充,可以实现细胞内多荧光标记,提供更理想的FRET荧光对。经随机突变和定点突变等方法改建获得的红荧光蛋白变种显示出更高的荧光强度,成熟时间也更短。目前应用较多的是来源于香菇珊瑚(Discosomasp.)的红荧光蛋白DsRed。     

含Dsred类似生色团的红色荧光蛋白的发展及应用

自从绿色荧光蛋白(GFP)被发现以来,荧光蛋白在生物医学领域已经成为一种重要的荧光成像工具．随着红色荧光蛋白DsRed的出现,各种优化的DsRed突变体和远红荧光蛋白也不断涌现．其中荧光蛋白生色团的形成机制对改建更优的荧光蛋白变种影响很大,对于红色荧光蛋白而言,大多数的红色荧光蛋白的生色团类型为DsRed类似生色团,在此基础上又出现了Far-red DsRed类似生色团．目前,含DsRed类似生色团的荧光蛋白主要有单体红色荧光蛋白、光转换荧光蛋白、斯托克斯红移蛋白、荧光计时器等．这些优化的荧光蛋白作为分子探针可以实现对活细胞、细胞器或胞内分子的时空标记和追踪,已经在生物工程学、细胞生物学、基础医学领域得到广泛应用．本文综述了含DsRed类似生色团的荧光蛋白的研究进展及其应用,以及由此发展起来的远红荧光蛋白在活体显微成像技术中的应用,并展望了荧光探针技术研究的新方向．     

Evolutionary and functional diversity of green fluorescent proteins in cephalochordates

Green fluorescent protein (GFP) has been widely used as a molecular marker in modern biological research. Before the recent report of one GFP gene in Branchiostoma floridae, GFP family members were cloned only from other two groups of species: Cnidaria and Copepoda. Here we describe the complete GFP gene repertoire of B. floridae which includes 13 functional genes and 2 pseudogenes, representing the largest GFP family found so far. Coupling with nine other GFP sequences from another two species of genus Branchiostoma and the sequences from Cnidaria and Copepoda, we made a deep-level phylogenetic analysis for GFP genes in cephalochordates and found: 1) GFP genes have experienced a divergent evolution in cephalochordates; 2) all amphioxus GFP genes form four main clades on the tree which had diverged before the radiation of the last common ancestor of all extant cephalochordates; 3) GFP genes in amphioxus shared a common ancestor with that in Copepoda rather than being derived from horizontal gene transfer, which indicates that our ancestor was derived from a fluorescent organism and lost this ability after its separation from Cephalochordata, and also makes GFP a rare gene which has a rather unusual evolutionary path. In addition, we also provided evidence indicating that GFP genes have evolved divergent functions by specializing their expression profile, and different fluorescent spectra by changing their emission peaks. These findings spark two interesting issues: what are GFP in vivo functions in cephalochordates and why they are lost in other examined deuterostomes?     

The 2.1A crystal structure of the far-red fluorescent protein HcRed: inherent conformational flexibility of the chromophore

We have determined the crystal structure of HcRed, a far-red fluorescent protein isolated from Heteractis crispa, to 2.1A resolution. HcRed was observed to form a dimer, in contrast to the monomeric form of green fluorescent protein (GFP) or the tetrameric forms of the GFP-like proteins (eqFP611, Rtms5 and DsRed). Unlike the well-defined chromophore conformation observed in GFP and the GFP-like proteins, the HcRed chromophore was observed to be considerably mobile. Within the HcRed structure, the cyclic tripeptide chromophore, Glu(64)-Tyr(65)-Gly(66), was observed to adopt both a cis coplanar and a trans non-coplanar conformation. As a result of these two conformations, the hydroxyphenyl moiety of the chromophore makes distinct interactions within the interior of the beta-can. These data together with a quantum chemical model of the chromophore, suggest the cis coplanar conformation to be consistent with the fluorescent properties of HcRed, and the trans non-coplanar conformation to be consistent with non-fluorescent properties of hcCP, the chromoprotein parent of HcRed. Moreover, within the GFP-like family, it appears that where conformational freedom is permissible then flexibility in the chromophore conformation is possible.     

Fluorescent proteins as a toolkit for in vivo imaging

Green fluorescent protein (GFP) from the jellyfish Aequorea victoria, and its mutant variants, are the only fully genetically encoded fluorescent probes available and they have proved to be excellent tools for labeling living specimens. Since 1999, numerous GFP homologues have been discovered in Anthozoa, Hydrozoa and Copepoda species, demonstrating the broad evolutionary and spectral diversity of this protein family. Mutagenic studies gave rise to diversified and optimized variants of fluorescent proteins, which have never been encountered in nature. This article gives an overview of the GFP-like proteins developed to date and their most common applications to study living specimens using fluorescence microscopy.     

The molecular properties and applications of Anthozoa fluorescent proteins and chromoproteins

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its fluorescent homologs from Anthozoa corals have become invaluable tools for in vivo imaging of cells and tissues. Despite spectral and chromophore diversity, about 100 cloned members of the GFP-like protein family possess common structural, biochemical and photophysical features. Anthozoa GFP-like proteins are available in colors and properties unlike those of A. victoria GFP variants and thus provide powerful new fluorophores for molecular labeling and intracellular detection. Although Anthozoa GFP-like proteins provide some advantages over GFP, they also have certain drawbacks, such as obligate oligomerization and slow or incomplete fluorescence maturation. In the past few years, effective approaches for eliminating some of these limitations have been described. In addition, several Anthozoa GFP-like proteins have been developed into novel imaging agents, such as monomeric red and dimeric far-red fluorescent proteins, fluorescent timers and photoconvertible fluorescent labels. Future studies on the structure of this diverse set of proteins will further enhance their use in animal tissues and as intracellular biosensors.     

Fluorescent proteins: physical-chemical properties and application in cell biology

Green fluorescent protein (GFP) from jellyfish Aequorea victoria is the most extensively studied and widely used in cell biology protein. At present novel naturally occurring GFP-like proteins have been discovered and enhanced mutants of Aequorea GFP have been created. These mutants differ from wild-type GFP by stability, value of quantum yield, absorption and fluorescence spectra position and photochemical properties. GFP-like proteins are the fast growing family. This review is an attempt to characterize the main groups of GFP-like proteins, describe their structure and mechanisms of chromophore formation and summarize the main trends of their utilization as markers and biosensors in cell and molecular biology.     

A new,long-wavelength borondipyrromethene sphingosine for studying sphingolipid dynamics in live cells

Sphingolipids function as cell membrane components and as signaling molecules that regulate critical cellular processes. To study unacylated and acylated sphingolipids in cells with fluorescence microscopy, the fluorophore in the analog must be located within the sphingoid backbone and not the N-acyl fatty acid side chain. Although such fluorescent sphingosine analogs have been reported, they either require UV excitation or their emission overlaps with that of the most common protein label, green fluorescent protein (GFP). We report the synthesis and use of a new fluorescent sphingolipid analog, borondipyrromethene (BODIPY) 540 sphingosine, which has an excitation maximum at 540 nm and emission that permits its visualization in parallel with GFP. Mammalian cells readily metabolized BODIPY 540 sphingosine to more complex fluorescent sphingolipids, and subsequently degraded these fluorescent sphingolipids via the native sphingolipid catabolism pathway. Visualization of BODIPY 540 fluorescence in parallel with GFP-labeled organelle-specific proteins showed the BODIPY 540 sphingosine metabolites were transported through the secretory pathway and were transiently located within lysosomes, mitochondria, and the nucleus. The reported method for using BODIPY 540 sphingosine to visualize sphingolipids in parallel with GFP-labeled proteins within living cells may permit new insight into sphingolipid transport, metabolism, and signaling.     

Denaturation studies reveal significant differences between GFP and blue fluorescent protein

Green fluorescent protein (GFP) is an unusually stable fluorescent protein that belongs to a family of related auto-fluorescent proteins (AFPs). These AFPs have been generated from jellyfish GFP by mutating the amino acids in the chromophore or its vicinity. Variants that emit light in the blue region (Blue Fluorescent Protein, BFP), red region, or yellow region are readily available and are widely used in diverse applications. Previously, we had used fluorescence spectroscopy to study the effect of pH on the denaturation of GFP with SDS, urea, and heat. Surprisingly, we found that SDS, urea or heat, did not have any significant effect on the fluorescence of GFP at pH 7.5 or 8.5, however, at pH 6.5, the protein lost all fluorescence within a very short period of time. These results suggested that GFP undergoes a structural/stability shift between pH 6.5 and 7.5, with the GFP structure at pH 6.5 being very sensitive to denaturation by SDS, urea, and heat. In the present study, we wanted to explore whether the stability or structure of the closely related BFP is also pH dependent. As expected, we found heat-induced denaturation and renaturation of BFP to be pH dependent, very much like GFP. However, when exposed to other denaturants like urea/heat or SDS we found BFP to behave very differently than GFP. Unlike GFP, which at pH 8.5 and 7.5 is very resistant to SDS-induced denaturation, BFP readily lost about 20% of its fluorescence at pH 8.5 and about 60% fluorescence at pH 7.5. Also, our denaturation and renaturation studies show that under certain conditions, BFP is more stable than GFP, such that under conditions where GFP is completely denatured, BFP still retained significant fluorescence. Taken together, our preliminary results show that despite being very similar in both amino acid sequences and overall structures, there may be subtle and important structural/conformational differences between BFP and GFP.     

Use of Green Fluorescent Protein (GFP) and Its Homologs for in vivo Protein Motility Studies

Green fluorescent protein (GFP) and its homologs are widely used as fluorescent markers of gene expression and for determination of protein localization and motility in living cells. In particular, based on GFP and GFP-like proteins a number of techniques have been developed that can be used either to estimate protein mobility in living cells, or to introduce a distinctive fluorescent signal in order to track the movement of labeled molecules directly. Considerable progress in the development of such technologies in the last two or three years motivates us to reevaluate the present scope of biotechnological instruments in studies of protein movement in cells.     

Development of Lactococcus lactis encoding fluorescent proteins,GFP, mCherry and iRFP regulated by the nisin-controlled gene expression system

Fluorescent proteins are useful reporter molecules for a variety of biological systems. We present an alternative strategy for cloning reporter genes that are regulated by the nisin-controlled gene expression (NICE) system. Lactoccocus lactis was genetically engineered to express green fluorescent protein (GFP), mCherry or near-infrared fluorescent protein (iRFP). The reporter gene sequences were optimized to be expressed by L. lactis using inducible promoter pNis within the pNZ8048 vector. Expression of constructions that carry mCherry or GFP was observed by fluorescence microscopy 2 h after induction with nisin. Expression of iRFP was evaluated at 700 nm using an infrared scanner; cultures induced for 6 h showed greater iRFP expression than non-induced cultures or those expressing GFP. We demonstrated that L. lactis can express efficiently GFP, mCherry and iRFP fluorescent proteins using an inducible expression system. These strains will be useful for live cell imaging studies in vitro or for imaging studies in vivo in the case of iRFP.     

Development of transgenic fish for ornamental and bioreactor by strong expression of fluorescent proteins in the skeletal muscle

In the present study, new applications of the transgenic technology in developing novel varieties of ornamental fish and bioreactor fish were explored in a model fish, the zebrafish (Danio rerio). Three "living color" fluorescent proteins, green fluorescent protein (GFP), yellow fluorescent protein (YFP), and red fluorescent protein (RFP or dsRed), were expressed under a strong muscle-specific mylz2 promoter in stable lines of transgenic zebrafish. These transgenic zebrafish display vivid fluorescent colors (green, red, yellow, or orange) visible to unaided eyes under both daylight and ultraviolet light in the dark. The level of foreign protein expression is estimated between 3% and 17% of total muscle proteins, equivalent to 4.8-27.2mg/g wet muscle tissue. Thus, the fish muscle may be explored as another useful bioreactor system for production of recombinant proteins. In spite of the high level of foreign protein expression, the expression of endogenous mylz2 mRNAs was not negatively affected. Furthermore, compared to the wild-type fish, these fluorescent transgenic fish have no advantage in survival and reproduction.