A photostable monomeric superfolder green fluorescent protein

The green fluorescent protein (GFP) from Aequorea victoria has been engineered extensively in the past to generate variants suitable for protein tagging. Early efforts produced the enhanced variant EGFP and its monomeric derivative mEGFP, which have useful photophysical properties, as well as superfolder GFP, which folds efficiently under adverse conditions. We previously generated msGFP, a monomeric superfolder derivative of EGFP. Unfortunately, compared to EGFP, msGFP and other superfolder GFP variants show faster photobleaching. We now describe msGFP2, which retains monomeric superfolder properties while being as photostable as EGFP. msGFP2 contains modified N‐ and C‐terminal peptides that are expected to reduce nonspecific interactions. Compared to EGFP and mEGFP, msGFP2 is less prone to disturbing the functions of certain partner proteins. For general‐purpose protein tagging, msGFP2 may be the best available derivative of A. victoria GFP.     

Novel fluorescent protein from Discosoma coral and its mutants possesses a unique far-red fluorescence

A novel gene for advanced red-shifted protein with an emission maximum at 593 nm was cloned from Discosoma coral. The protein, named dsFP593, is highly homologous to the recently described GFP-like protein drFP583 with an emission maximum at 583 nm. Using the remarkable similarity of the drFP583 and dsFP593 genes, we performed a 'shuffling' procedure to generate a pool of mutants consisting of various combinations of parts of both genes. One 'hybrid gene' was chosen for subsequent random mutagenesis, which resulted in a mutant variant with a uniquely red-shifted emission maximum at 616 nm.     

A site-specific bifunctional protein labeling system for affinity and fluorescent analysis

Most covalent protein labeling schemes require a choice between visual and affinity properties, requiring the use of multiple fusion systems where both attributes are needed. While not disruptive at the single experiment level, this detail becomes critical when addressing high-throughput experimentation. Here we develop a uniform site-specific protein tag for use in both fluorescent and affinity screening. Covalent protein tagging with a stilbene reporter via promiscuous phosphopantetheinyltransferase (PPTase) modification enables a switchable, antibody-elicited fluorescent response in solution or on affinity resin. For demonstration purposes, VibB, a natural fusion protein harboring a carrier protein domain, was labeled with a stilbene tag through PPTase modification with a stilbene-labeled coenzyme A analogue. Analysis of the resulting stilbene-tagged VibB was accomplished by fluorescent and Western blot analysis with anti-stilbene monoclonal antibody EP2-19G2. The illustration of this method for general application to fusion protein analysis offers a dual role in assisting both solution-based fluorescent analysis and surface-based affinity detection and purification.     

The fluorescent monomeric protein Kusabira Orange. Pressure effect on its structure and stability

The structure and stability of the fluorescent protein monomeric Kusabira Orange (mKO), a GFP-like protein, was studied under different pressure levels and in different chemical environments. At different pH values (between pH 7.4 and pH 4.0) and under a pressure up to 600 MPa (at 25 °C), mKO did not show significant fluorescence spectral changes, indicating a structural stability of the protein. In more extreme chemical conditions (at pH 4.0 in the presence of 0.8 M guanidine hydrochloride), a marked reduction of mKO fluorescence intensity emission was observed at pressures above 300 MPa. This fluorescence emission quenching may be due to the loss of the intermolecular bonds and, consequently, to the destructuration of the mKO chromophore structure. Since the electrostatic and hydrophobic interactions as well as the salt bridges present in proteins are usually perturbed under high pressure, the reduction of mKO fluorescence intensity emission is associated to the perturbation of the protein salt bridges network.     

A brilliant monomeric red fluorescent protein to visualize cytoskeleton dynamics in Dictyostelium

Red fluorescent proteins (RFPs) combined with GFP are attractive probes for double-fluorescence labeling of proteins in live cells. However, the application of these proteins is restrained by stable oligomer formation and by their weak fluorescence in vivo. Previous attempts to eliminate these problems by mutagenesis of RFP from Discosoma (DsRed) resulted in the monomeric mRFP1 and in the tetrameric RedStar RFP, which is distinguished by its enhanced fluorescence in vivo. Based on these mutations, we have generated an enhanced monomeric RFP, mRFPmars, and report its spectral properties. Together with green fluorescent labels, we used mRFPmars to visualize filamentous actin structures and microtubules in Dictyostelium cells. This enhanced RFP proved to be suitable to monitor the dynamics of cytoskeletal proteins in cell motility, mitosis, and endocytosis using dual-wavelength fluorescence microscopy.     

Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein

Fluorescent proteins are genetically encoded, easily imaged reporters crucial in biology and biotechnology. When a protein is tagged by fusion to a fluorescent protein, interactions between fluorescent proteins can undesirably disturb targeting or function. Unfortunately, all wild-type yellow-to-red fluorescent proteins reported so far are obligately tetrameric and often toxic or disruptive. The first true monomer was mRFP1, derived from the Discosoma sp. fluorescent protein "DsRed" by directed evolution first to increase the speed of maturation, then to break each subunit interface while restoring fluorescence, which cumulatively required 33 substitutions. Although mRFP1 has already proven widely useful, several properties could bear improvement and more colors would be welcome. We report the next generation of monomers. The latest red version matures more completely, is more tolerant of N-terminal fusions and is over tenfold more photostable than mRFP1. Three monomers with distinguishable hues from yellow-orange to red-orange have higher quantum efficiencies.     

Optimized synthesis of functionalized fluorescent oligodeoxynucleotides for protein labeling

Phthalimido-alkanol solid supports were rapidly prepared from solid supported phthalic anhydride and amino alcohol condensation induced by microwaves. These supports were used to synthesize 5'-fluorescent 3'-aminoalkyl oligodeoxynucleotides allowing a two-step deprotection necessary to avoid aminolink alkylation. After conversion into an NHS derivative using dissuccinimidyl suberate and an optimized isolation, they were conjugated with a protein.     

Methodology of reversible protein labeling for ratiometric fluorescent measurement

The first fluorescent labeling technology, which can induce not only an increase in the fluorescence intensity but also a shift in the fluorescence spectrum, has been developed for "ratiometric" measurements for a protein by utilizing a newly designed "field-sensitive" fluorescent probe and its corresponding unique amino acid tag.     

Modeling spectral tuning in monomeric teal fluorescent protein mTFP1

We present results of theoretical studies of the variants of the monomeric teal fluorescent protein from Clavularia coral (mTFP1) which present promising members from the GFP family. Predictions of quantum chemical approaches including density functional theory and semiempirical approximations are presented for the model systems which mimic the chromophores in different environments. We describe the excitation energy spectrum of the cyan mTFP1 fluorescent protein with the original chromophore and with chromophore mutants Tyr67His and Tyr67Trp.     

Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging

The arsenal of engineered variants of the GFP [green FP (fluorescent protein)] from Aequorea jellyfish provides researchers with a powerful set of tools for use in biochemical and cell biology research. The recent discovery of diverse FPs in Anthozoa coral species has provided protein engineers with an abundance of alternative progenitor FPs from which improved variants that complement or supersede existing Aequorea GFP variants could be derived. Here, we report the engineering of the first monomeric version of the tetrameric CFP (cyan FP) cFP484 from Clavularia coral. Starting from a designed synthetic gene library with mammalian codon preferences, we identified dimeric cFP484 variants with fluorescent brightness significantly greater than the wild-type protein. Following incorporation of dimer-breaking mutations and extensive directed evolution with selection for blue-shifted emission, high fluorescent brightness and photostability, we arrived at an optimized variant that we have named mTFP1 [monomeric TFP1 (teal FP 1)]. The new mTFP1 is one of the brightest and most photostable FPs reported to date. In addition, the fluorescence is insensitive to physiologically relevant pH changes and the fluorescence lifetime decay is best fitted as a single exponential. The 1.19 A crystal structure (1 A=0.1 nm) of mTFP1 confirms the monomeric structure and reveals an unusually distorted chromophore conformation. As we experimentally demonstrate, the high quantum yield of mTFP1 (0.85) makes it particularly suitable as a replacement for ECFP (enhanced CFP) or Cerulean as a FRET (fluorescence resonance energy transfer) donor to either a yellow or orange FP acceptor.     

A recombinant replication-competent hepatitis C virus expressing Azami-Green, a bright green-emitting fluorescent protein, suitable for visualization of infected cells

The hepatitis C virus (HCV) production system consists of transfecting the human hepatoma cell line Huh7 with genomic HCV RNA (JFH1). To monitor HCV replication by fluorescence microscopy, we constructed a recombinant HCV clone expressing Azami-Green (mAG), a bright green fluorescent protein, by inserting the mAG gene into the nonstructural protein 5A (NS5A) gene; the resultant clone was designated JFH1-hmAG. The Huh-7.5.1 (a subclone of Huh7) cells transfected with JFH1-hmAG RNA were found to produce cytoplasmic NS5A-mAG, as readily visualized by fluorescence microscopy, and infectious virus, as assayed with the culture supernatant, indicating that JFH1-hmAG is infectious and replication-competent. Furthermore, the replication of this virus was inhibited by interferon alpha in a dose-dependent manner. These results suggest that JFH1-hmAG is useful for studying HCV life cycle and the mechanism of interferon’s anti-HCV action and for screening and testing new anti-HCV drugs.     

Ubiquitous expression of the monomeric red fluorescent protein mcherry in transgenic mice

The use of the green fluorescent protein (GFP) to label specific cell types and track gene expression in animal models, such as mice, has evolved to become an essential tool in biological research. Transgenic animals expressing genes of interest linked to GFP, either as a fusion protein or transcribed from an internal ribosomal entry site (IRES) are widely used. Enhanced GFP (eGFP) is the most common form of GFP used for such applications. However, a red fluorescent protein (RFP) would be highly desirable for use in dual‐labeling applications with GFP derived fluorescent proteins, and for deep in vivo imaging of tissues. Recently, a new generation of monomeric (m)RFPs, such as monomeric (m)Cherry, has been developed that are potentially useful experimentally. mCherry exhibits brighter fluorescence, matures more rapidly, has a higher tolerance for N‐terminal fusion proteins, and is more photostable compared with its predecessor mRFP1. mRFP1 itself was the first true monomer derived from its ancestor DsRed, an obligate tetramer in vivo. Here, we report the successful generation of a transgenic mouse line expressing mCherry as a fluorescent marker, driven by the ubiquitin‐C promoter. mCherry is expressed in almost all tissues analyzed including pre‐ and post‐implantation stage embryos, and white blood cells. No expression was detected in erythrocytes and thrombocytes. Importantly, we did not encounter any changes in normal development, general physiology, or reproduction. mCherry is spectrally and genetically distinct from eGFP and, therefore, serves as an excellent red fluorescent marker alone or in combination with eGFP for labelling transgenic animals. genesis 48:723–729, 2010. © 2010 Wiley‐Liss, Inc.     

A biological cell labeling technique and its use in expermental embryology

A cell-labeling technique is proposed, based on structural differences between the interphase nucleus of two species of birds, the japanese quail (Coturnix coturnix japonica) and the chick (Gallus gallus). The quail nucleus shows large heterochromatic masses associated with the nucleolar RNA. In the chick, on the contrary, the arrangement of chromatin during the interphase fits the general pattern observed in vertebrate cells: DNA is rather uniformly distributed in the nucleoplasm, and the quantity of nucleolus-associated chromatin is not significant. It is possible to distinguish the cells of the two species in histological sections stained by the Feulgen Rossenbeck reaction, and also at the electron microscope level in sections involving the nucleolus. Thus quail cells can be used as “natural markers” to study various embryological problems such as those related to intercellular interactions and cell migration during ontogeny.     

枯草杆菌ylyA基因的绿色荧光蛋白标记

[目的]本研究对枯草杆菌ylyA基因进行荧光标记以便对其产物YlyA在菌体中的位置进行初步观察.[方法]以不同菌株基因组DNA为模板,对ylyA基因进行PCR扩增和序列分析;重新设计引物扩增全长的ylyA并将其克隆到载体pSG1729中,形成gfpmut1-ylyA融合而构建重组载体pNG426;将pNG426转化枯草杆菌168菌株,双交换使gfpmut1-ylyA插入染色体的amyE位点,用碘染色法和菌落PCR对阳性转化子BS363进行鉴定.NA固体培养基上生长的BS363经0.5%木糖诱导表达后,利用表面荧光显微镜技术进行观察.[结果]通过对多个PCR产物的序列分析确定了ylyA基因的正确序列以及正确的翻译起始位点;成功将重组载体pNG426转化枯草杆菌得到了BS363菌株;荧光检测结果表明GFP标记的YlyA分布于菌体的外周,在位置上靠近细胞膜并与之平行排列.[结论]生长缓慢的BS363菌体,在0.5%木糖诱导下产生的荧光标记YlyA蛋白分布在细胞外周,可能在膜生物学中发挥作用.     

A fluorescent variant of a protein from the stony coral Montipora facilitates dual-color single-laser fluorescence cross-correlation spectroscopy

Dual-color fluorescence cross-correlation spectroscopy (FCCS) is a promising technique for quantifying protein-protein interactions. In this technique, two different fluorescent labels are excited and detected simultaneously within a common measurement volume. Difficulties in aligning two laser lines and emission crossover between the two fluorophores, however, make this technique complex. To overcome these limitations, we developed a fluorescent protein with a large Stokes shift. This protein, named Keima, absorbs and emits light maximally at 440 nm and 620 nm, respectively. Combining a monomeric version of Keima with cyan fluorescent protein allowed dual-color FCCS with a single 458-nm laser line and complete separation of the fluorescent protein emissions. This FCCS approach enabled sensitive detection of proteolysis by caspase-3 and the association of calmodulin with calmodulin-dependent enzymes. In addition, Keima and a spectral variant that emits maximally at 570 nm might facilitate simultaneous multicolor imaging with single-wavelength excitation.     

Brightness of yellow fluorescent protein from coral (zFP538) depends on aggregation

The yellow fluorescent protein from coral (zFP538) forms aggregates in water solutions. According to dynamic light scattering and gel filtration data, the aggregation number is approximately 1000-10000 at pH 8-9 and protein concentration 1 mg/mL. Gel filtration demonstrated that dissociation of the aggregates takes place upon dilution, and the molecular weight of the aggregates decreases with pH. Atomic force microscopy (AFM) and near-field scanning optical microscopy (NSOM) were used to obtain images of zFP538 in the solid state. It was shown that protein films are comprised of fluorescent ellipsoidal granules with a 50-300 nm major axis and a 30-130 nm minor axis. The dependence of zFP538 fluorescence on protein concentration between 1.2 x 10(-)(9) and 5.5 x 10(-)(7) M can be divided in two linear regions with different slopes indicating the existence of at least two different forms of zFP538. The fluorescence of zFP538 decreases with time upon acidification, and the decrease depends on pH and protein concentration. Between pH 3.5 and pH 5.5, relative residual fluorescence is higher for concentrated zFP538 solutions (about 10(-)(6) M) as compared with diluted ones (10(-)(7) M and below). Aggregation makes zFP538 more stable against fluorescence quenching upon acidification: the decrease in zFP538 fluorescence at protein concentration 1 mg/mL is completely reversible, unlike that observed for less concentrated solutions. This phenomenon may be due to the decrease in the freedom of chromophore mobility in zFP538 aggregates.     

A fluorescent dual labeling technique for the quantitative measurement of reduced and oxidized protein thiols in tissue samples

Oxidative stress can result in the reversible oxidation of protein thiols. Because the activity of numerous proteins is sensitive to thiol oxidation, this has the potential to affect many cellular functions. We describe a highly sensitive, quantitative labeling technique that measures global and specific protein thiol oxidative state in skeletal muscle tissue. The technique involves labeling the reduced and oxidized protein thiols with different fluorescent dyes. The resulting sample is assayed using a 96-well plate fluorimeter, or individual protein bands are separated using SDS-PAGE. We show that artifactual oxidation during sample preparation and analysis has the potential to confound results, and techniques to prevent this are described. We tested the technique by analyzing the muscles of mdx and c57 mice and found that the muscles of mdx mice were significantly (p<0.05) more oxidized (13.1±1.5% oxidized thiols) than those of c57 mice (8.9±0.7% oxidized thiols). This technique provides an effective means to measure the extent to which oxidative stress affects the oxidation of protein thiols in biological tissues.     

Crystallographic evidence for water-assisted photo-induced peptide cleavage in the stony coral fluorescent protein Kaede

A coral fluorescent protein from Trachyphyllia geoffroyi, Kaede, possesses a tripeptide of His62-Tyr63-Gly64, which forms a chromophore with green fluorescence. This chromophore's fluorescence turns red following UV light irradiation. We have previously shown that such photoconversion is achieved by a formal beta-elimination reaction, which results in a cleavage of the peptide bond found between the amide nitrogen and the alpha-carbon at His62. However, the stereochemical arrangement of the chromophore and the precise structural basis for this reaction mechanism previously remained unknown. Here, we report the crystal structures of the green and red form of Kaede at 1.4 A and 1.6 A resolutions, respectively. Our structures depict the cleaved peptide bond in the red form. The chromophore conformations both in the green and red forms are similar, except a well-defined water molecule in the proximity of the His62 imidazole ring in the green form. We propose a molecular mechanism for green-to-red photoconversion, which is assisted by the water molecule.     

Reliable use of green fluorescent protein in fluorescent pseudomonads.

When fluorescent pseudomonads are cultured on standard solid media under iron limiting conditions, they produce fluorescent, pigmented iron collating agents (siderophores). Siderophores can be readily identified by strong fluorescence seen under UV/blue light. The application of the eukaryotic green fluorescent protein (GFP) as a bacterial marker in microbial ecology is increasingly being used, particularly as it is a powerful method for non-destructive monitoring in situ. As gfp expressing bacteria have to be detected under UV/blue light, the fluorescence of siderophore-producing Pseudomonas spp. masks normal levels of GFP fluorescence when colonies are viewed on standard bacterial agar. Here, we describe a simple but effective way of identifying gfp-expressing Pseudomonas fluorescens using media supplemented with 0.45 mM FeSO(4).7H(2)O. This is of relevance for the screening of insertion libraries and in the application of GFP transposons as promoter probes.