Photoconversion of some fluorescent markers to a diaminobenzidine product

Retinal whole mounts, brain sections, and astrocyte cultures were labeled with various fluorescent markers. Tissues or cells were then irradiated by light in the presence of diaminobenzidine. Irradiation initiated a reaction in which specific fluorescent labeling was replaced by an insoluble diaminobenzidine product. The diaminobenzidine product is more stable than the original fluorescent labeling and can be processed for electron microscopy. In some cases, the reaction product reveals cellular detail that cannot be resolved in the fluorescent labeling. The 10 fluorescent markers tested have widely differing structures, span a broad range of wavelengths, and label several different cellular elements. The photoconversion reaction was successful with all markers and tissues tested.     

Photoconversion of DAPI and Hoechst dyes to green and red-emitting forms after exposure to UV excitation

The fluorescent dye 4′-6-Diamidino-2-phenylindole (DAPI) is frequently used in fluorescence microscopy as a chromosome and nuclear stain because of its high specificity for DNA. Normally, DAPI bound to DNA is maximally excited by ultraviolet (UV) light at 358 nm, and emits maximally in the blue range, at 461 nm. Hoechst dyes 33258 and 33342 have similar excitation and emission spectra and are also used to stain nuclei and chromosomes. It has been reported that exposure to UV can convert DAPI and Hoechst dyes to forms that are excited by blue light and emit green fluorescence, potentially confusing the interpretation of experiments that use more than one fluorochrome. The work reported here shows that these dyes can also be converted to forms that are excited by green light and emit red fluorescence. This was observed both in whole tissues and in mitotic chromosome spreads, and could be seen with less than 10-s exposure to UV. In most cases, the red form of fluorescence was more intense than the green form. Therefore, appropriate care should be exercised when examining tissues, capturing images, or interpreting images in experiments that use these dyes in combination with other fluorochromes.     

Expression and characterization of a redox-sensing green fluorescent protein (reduction-oxidation-sensitive green fluorescent protein) in Arabidopsis

Arabidopsis (Arabidopsis thaliana) was transformed with a redox-sensing green fluorescent protein (reduction-oxidation-sensitive green fluorescent protein [roGFP]), with expression targeted to either the cytoplasm or to the mitochondria. Both the mitochondrial and cytosolic forms are oxidation-reduction sensitive, as indicated by a change in the ratio of 510 nm light (green light) emitted following alternating illumination with 410 and 474 nm light. The 410/474 fluorescence ratio is related to the redox potential (in millivolts) of the organelle, cell, or tissue. Both forms of roGFP can be reduced with dithiothreitol and oxidized with hydrogen peroxide. The average resting redox potentials for roots are -318 mV for the cytoplasm and -362 mV for the mitochondria. The elongation zone of the Arabidopsis root has a more oxidized redox status than either the root cap or meristem. Mitochondria are much better than the cytoplasm, as a whole, at buffering changes in redox. The data show that roGFP is redox sensitive in plant cells and that this sensor makes it possible to monitor, in real time, dynamic changes in redox in vivo.     

Post-translational targeting of a tail-anchored green fluorescent protein to the endolpasmic reticulum

Microsomal aldehyde dehydrogenase (msALDH) is a tail-anchored protein localized to the cytoplasmic face of the endoplasmic reticulum (ER). The carboxyl-terminal 35 amino acids of msALDH possess ER-targeting sequences in addition to a hydrophobic membrane-spanning domain. To study the mechanism for ER targeting of this protein in vivo, we took advantage of a green fluorescent protein-msALDH fusion protein containing the last 35 amino acids of msALDH [GFPALDH(35)]. When expressed from cDNA in COS-7 cells, the fusion protein was localized to the ER. We then prepared a recombinant fusion protein and injected it into the cytoplasm of COS-7 cells. The injected protein was correctly localized to the ER after a 30-min incubation at 37 degrees C. However, a recombinant fusion protein that contained only the transmembrane domain of msALDH failed to be targeted to the ER. When the assay was carried out at 4 degrees C, the recombinant GFPALDH(35) remained in the cytoplasm. Moreover, incubation of COS-7 cells under conditions of ATP depletion resulted in the cytoplasmic distribution of the injected protein. These results indicate that GFPALDH(35) is targeted to the ER post-translationally via an ATP-dependent pathway. This microinjection system worked effectively in different mammalian cell types, suggesting a common mechanism for ER targeting of the tail-anchored protein.     

GFP在绿色荧光裸鼠不同组织中的表达及分析

目的研究外源绿色荧光蛋白（green fluorescent protein,简称GFP）基因在BALB/c绿色荧光裸鼠主要器官组织中的表达及其差异。方法小动物成像系统和RT-PCR方法检测GFP的组织分布以及荧光表达水平情况。结果经活体荧光影像系统观察及PCR方法检测发现GFP可以在裸鼠多个器官组织中表达,其中在胰腺、心脏、全脑、皮肤、睾丸中表达量较高。结论外源绿色荧光蛋白可以在模型动物体内成功表达且稳定遗传,其中在胰腺组织中高表达。     

Purification of green fluorescent protein using a two-intein system

A two-intein purification system was developed for the affinity purification of GFPmut3*, a mutant of green fluorescent protein. The GFPmut3* was sandwiched between two self-cleaving inteins. This approach avoided the loss of the target protein which may result from in vivo cleavage of a single intein tag. The presence of N- and C-terminal chitin-binding domains allowed the affinity purification by a single-affinity chitin column. After the fusion protein was expressed and immobilized on the affinity column, self-cleavage of the inteins was sequentially induced to release the GFPmut3*. The yield was 2.41 mg from 1 l of bacterial culture. Assays revealed that the purity was up to 98% of the total protein. The fluorescence and circular dichroism spectrum of GFPmut3* demonstrated that the purified protein retains the correctly folded structure and function.     

Engineering and characterization of a superfolder green fluorescent protein

Existing variants of green fluorescent protein (GFP) often misfold when expressed as fusions with other proteins. We have generated a robustly folded version of GFP, called 'superfolder' GFP, that folds well even when fused to poorly folded polypeptides. Compared to 'folding reporter' GFP, a folding-enhanced GFP containing the 'cycle-3' mutations and the 'enhanced GFP' mutations F64L and S65T, superfolder GFP shows improved tolerance of circular permutation, greater resistance to chemical denaturants and improved folding kinetics. The fluorescence of Escherichia coli cells expressing each of eighteen proteins from Pyrobaculum aerophilum as fusions with superfolder GFP was proportional to total protein expression. In contrast, fluorescence of folding reporter GFP fusion proteins was strongly correlated with the productive folding yield of the passenger protein. X-ray crystallographic structural analyses helped explain the enhanced folding of superfolder GFP relative to folding reporter GFP.     

Vibrational analysis of a solvated green fluorescent protein chromophore

Resonance Raman (RR) spectra of green fluorescent protein (GFP) model chromophores in solution have been simulated with the CASSCF/MM methodology. Although several reports on vibrational analysis of GFP model chromophores have been recently published, the RR spectra were simulated for the first time in explicit solution with the inclusion of the counterion, as these effects are crucial for unambiguously reproducing the vibrational band assignment in the anionic form of the GFP chromophore. This strategy allows for a one-to-one correspondence of the calculated vibrational modes to the observed RR bands, concerning both the location and intensity pattern. In addition, these simulations were complemented with total energy distribution calculations to aid in the unambiguous assignment of the measured spectra. The current study helps to clarify some of the previous RR bands assignments as well as producing some new assignment for the anionic form of GFP chromophore. The explicit solvent simulations and PCM-based calculations are compared to the measured spectra, and these results demonstrate that explicit solvent simulations provide better agreement with experiment, both in terms of vibrational frequencies and intensity distribution. Figure a Correlation of explicit hydration calculations (CASSCF/6-31G*/MM) for the HBI model chromophore and experimental RR data [21]; slope = 0.982, intercept = 27.210 and regression coefficient = 0.997. b Correlation of implicit PCM calculations (CASSCF/6-31G*) for the HBI model chromophore and experimental RR data [21], slope = 1.017, intercept = −48.838 and regression coefficient = 0.984     

Photoconversion of protochlorophyll to chlorophyll a in a mutant of Chlorella regularis

Dark-grown cells of a mutant strain of Chlorella regularis containedchlorophyll a and protochlorophyll, phytyl ester of protochlorophyllide.Under illumination, protochlorophyll was quantitatively anddirectly converted into chlorophyll a. The photoconversion wasdependent on light intensity and temperature and proceeded ina cell-free preparation. The pathway of chlorophyll formation found in the mutant cellsis entirely different from that from protochlorophyllide byway of chlorophyllide a, which is generally observed in greenplants. ¹Present address: Division of Biology, Medical College of Miyazaki,Miyazaki 889-16, Japan. ²Present address: Division of Environmental Biology, The NationalInstitute for Environmental Studies, Ibaragi 300-21, Japan. (Received October 24, 1975; )     

Targeting aequorin and green fluorescent protein to intracellular organelles

Two proteins of Aequorea victoria were molecularly engineered and produced in mammalian cells, in order to serve as specific reporters of subcellular microenvironments. Aequorin (AEQ), a Ca²⁺-sensitive photoprotein, was successfully targeted to three intracellular locations: cytosol, nucleus and mitochondria. The recombinant apoprotein, reconstituted into active AEQ by the addition of the prosthetic group to the culture medium, allows the direct measurement of [Ca²⁺] within those compartments, thus directly addressing questions of large biological interest. The same approach was utilized for the green fluorescent protein (GFP) for specific labelling, in vivo, of the various subcellular structures. GFP was targeted to mitochondria: the recombinant protein, strongly fluorescent in a highly reducing environment, provides a powerful tool for visualizing these organelles in living cells, and may represent the prototype of a new family of intracellularly targeted fluorescent probes.     

High expression of green fluorescent protein in Pichia pastoris leads to formation of fluorescent particles

Wild type gene for green fluorescent protein (GFP) was stably integrated into the Pichia pastoris genome and yielded an expression level of over 40% of total cellular protein. The high cytoplasmic concentration of fluorescent (properly folded and processed) GFP caused the formation of fluorescent spherical structures, which could be observed by fluorescence or confocal microscopy after controlled permeabilization of the yeast cells with 0.2% N-lauroyl sarcosine (NLS). Fluorescent GFP particles were also isolated after removal of the cell wall and found to be quite resistant to 0.2% N-lauroyl sarcosine. SDS-PAGE analysis of the isolated fluorescent particles revealed the presence of an 80 kDa protein (alcohol oxidase) and GFP (30%). We conclude that GFP is able to enter spontaneously into the peroxisomes and is inserted into densely packed layers of alcohol oxidase. Consequently, the formation of similar fluorescent particles can also be expected in other organisms when using high-level expression systems. As GFP is widely used in fusion with other proteins as a reporter for protein localization and for many other applications in biotechnology, care must be taken to avoid false interpretations of targeting or trafficking mechanisms inside the cells. In addition, when whole cells or cytoplasmic fractions are used for the quantitative determination of GFP levels, incorrect and misleading values of GFP could be obtained due to the formation of fluorescent particles containing material inside which is not available for fluorescence measurements.     

The green fluorescent protein as a marker to visualize plant mitochondria in vivo

To determine how to utilize the green fluorescent protein (GFP) as a marker for subcellular localization and as a label for plant mitochondria in vivo, transgenic suspension cells and tobacco plants expressing GFP with and without a mitochondrial localization signal were generated. The first GFP form used, GFP1, is easily observable in cells with low autofluorescence, such as suspension cells or trichomes, but masked in green tissue. For the visualization of GFP in cells and tissues with high autofluorescence, such as leaf, the use of a very strong promoter (35S35SAMV), a highly expressed modified mGFP4 coding region and a brighter mutant form of GFP (S65T) was necessary. Confocal or two-photon laser scanning microscopy reveal a distinct subcellular localization of the fluorescence in cells expressing GFP or coxIVGFP. In cells expressing untargeted GFP, fluorescence accumulates in the nucleoplasm but is also distributed throughout the cytoplasm. It is excluded from vacuoles, nucleoli and from round bodies that are likely to be leucoplasts. In contrast, fluorescence is localized specifically to mitochondria in cells expressing coxIVGFP fusion protein as shown by co-localization with a mitochondrial-specific dye. This permits the direct observation of mitochondria and mitochondrial movements in living plant cells and tissues throughout plant development. Three-dimensional reconstruction of individual cells can give additional information about the distribution and numbers of mitochondria.     

Engineering green fluorescent protein as a dual functional tag

A hexa-histidine (6 x His) sequence was inserted into a surface loop of the green fluorescent protein (GFP) to develop a dual functional GFP useful for both monitoring and purification of recombinant proteins. Two variants (GFP172 and GFP157), differentiated by the site of insertion of the 6xHis sequence, were developed and compared with a control variant (GFPHis) having the 6xHis sequence at its C-terminus. The variants were produced in Escherichia coli and purified using immobilized metal affinity chromatography (IMAC). The purification efficiencies by IMAC for all variants were found to be comparable. Purified GFP172 and GFP157 variants retained approximately 60% of the fluorescence compared to that of GFPHis. The reduction in the fluorescence intensity associated with GFP172 and GFP157 was attributed to the lower percentage of fluorescent GFP molecules in these variants. Nonetheless, the rates of fluorescence acquisition were found to be similar for all functional variants. Protein misfolding at an elevated temperature (37 degrees C) was found to be less profound for GFP172 than for GFP157. The dual functional properties of GFP172 were tested with maltose binding protein (MBP) as the fusion partner. The MBP-GFP172 fusion protein remained fluorescent and was purified from E. coli lysate as well as from spiked tobacco leaf extracts in a single-step IMAC. For the latter, a recovery yield of approximately 75% was achieved and MBP-GFP172 was found to coelute with a degraded product of the fusion protein at a ratio of about 4:1. The primary advantage of the chimeric GFP tag having an internal hexa-histidine sequence is that such a tag allows maximum flexibility for protein or peptide fusions since both N- and C-terminal ends of the GFP are available for fusion.     

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.     

Binding of chimeric metal-binding green fluorescent protein to lipid monolayer

Membrane-based bioanalytical devices for metal determination using green fluorescent protein as the sensor molecule may be a useful future biomimetic material. However, in order to develop such a device, it is necessary first to understand the interaction of the protein with lipid membranes. Thus we have investigated the interaction between chimeric cadmium-binding green fluorescent proteins (CdBPGFPs) and lipid monolayers, using a film-balance technique complemented with epifluorescence microscopy. The binding avidity was monitored from the surface pressure vs. area isotherms or from the measured increase in the lateral pressure upon injection of the chimeric CdBPGFPs beneath the lipid monolayer. Increased fluidization as well as expansion of the surface area were shown to depend on the concentration of the CdBPGFPs. The kinetics of the protein-induced increase in lateral pressure was found to be biphasic. The chimeric CdBPGFPs possessed high affinity to the 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) monolayer with a dissociation constant of K_d=10^–8M. Epifluorescence measurements showed that this affinity is due to the presence of the Cd-binding peptide, which caused the GFP to incorporate preferentially to the liquid phase and defect part of the rigid domain at low interfacial pressure. At high compression, the Cd-binding peptide could neither incorporate nor remain in the lipid core. However, specific orientation of the chimeric CdBPGFPs underneath the air–water interface was achieved, even under high surface pressure, when the proteins were applied to the metal-chelating lipid-containing surfaces. This specific binding could be controlled reversibly by the addition of metal ions or metal chelator. The reversible binding of the chimeric CdBPGFPs to metal-chelating lipids provided a potential approach for immobilization, orientation and lateral organization of a protein at the membrane interface. Furthermore, the feasibility of applying the chelator lipids for the codetermination of metal ions with specific ligands was also revealed. Our finding clearly demonstrates that a strong interaction, particularly with fluid lipid domains, could potentially be used for sensor development in the future.Abbreviations GFP green fluorescent protein - CdBPGFPs cadmium-binding green fluorescent protein - DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine - AAS atomic absorption spectrometry - Cd²⁺ cadmium (II) - Zn²⁺ zinc (II) - Cu²⁺ copper (II) - Ni²⁺ nickel (II) - E. coli Escherichia coli - NTA-DOGS 1,2-dioleoyl-sn-glycero-3-(N-(5-amino-1-carboxypentyl iminodiacetic acid) succinyl) - His6GFP hexahistidine green fluorescent protein - CdBP4GFP four-repeat cadmium-binding peptide green fluorescent protein - His6CdBP4GFP hexahistidine four-repeat cadmium-binding peptide green fluorescent protein - IMAC immobilized-metal-affinity chromatography - PBS phosphate-buffered saline - mN/m millinewton per metre - le liquid expanded - lc liquid condensed - PE phosphatidyl ethanolamine - PI phosphatidyl inositol - NTA nitrilotriacetic acid - EDTA ethylenediamine tetraacetic acid - RESA ring-infected erythrocyte surface antigen - CdBP cadmium-binding peptide     

Novel sequence-responding fluorescent oligoDNA probe bearing a silylated pyrene molecule

A novel fluorescent phosphoramidite derivative of dimethylsilylated pyrene was prepared and incorporated into oligoDNA. The fluorescent oligoDNA exhibited marked fluorescent signal upon binding to the fully matched complementary DNA strand, however, the signal was strongly quenched in the single-stranded form as well as in the duplex having mismatched base pair at the terminus of the duplex-forming region.     

Vibrations-determined properties of green fluorescent protein

The physicochemical characteristics of the green fluorescent protein (GFP), including the thermodynamic properties (entropy, enthalpy, Gibbs' free energy, heat capacity), normal mode vibrations, and atomic fluctuations, were investigated. The Gaussian 03 computational chemistry program was employed for normal mode analysis using the AMBER force field. The thermodynamic parameters and atomic fluctuations were then calculated from the vibrational eigenvalues (frequencies) and eigenvectors. The regions of highest rigidity were shown to be the beta-sheet barrel with the central alpha-helix, which bears the chromophore. The most flexible parts of the GFP molecule were the outlying loops that cover the top and bottom of the beta-barrel. This way, the balance between rigidity and flexibility is maintained, which is the optimal relationship for protein stability in terms of Gibbs' free energy. This dual-schemed structure satisfies the requirements for GFP function. In this sense, the structure of GFP resembles a nanoscale drum: a stiff cylinder with flexible vibrating end(s).