Blue Fluorescent cGMP Sensor for Multiparameter Fluorescence Imaging

Cyclic GMP (cGMP) regulates many physiological processes by cooperating with the other signaling molecules such as cyclic AMP (cAMP) and Ca²⁺. Genetically encoded sensors for cGMP have been developed based on fluorescence resonance energy transfer (FRET) between fluorescent proteins. However, to analyze the dynamic relationship among these second messengers, combined use of existing sensors in a single cell is inadequate because of the significant spectral overlaps. A single wavelength indicator is an effective alternative to avoid this problem, but color variants of a single fluorescent protein-based biosensor are limited. In this study, to construct a new color fluorescent sensor, we converted the FRET-based sensor into a single wavelength indicator using a dark FRET acceptor. We developed a blue fluorescent cGMP biosensor, which is spectrally compatible with a FRET-based cAMP sensor using cyan and yellow fluorescent proteins (CFP/YFP). We cotransfected them and loaded a red fluorescent probe for Ca²⁺ into cells, and accomplished triple-parameter fluorescence imaging of these cyclic nucleotides and Ca²⁺, confirming the applicability of this combination to individually monitor their dynamics in a single cell. This blue fluorescent sensor and the approach using this FRET pair would be useful for multiparameter fluorescence imaging to understand complex signal transduction networks.     

Vibrio azureus emits blue-shifted light via an accessory blue fluorescent protein

Luminous marine bacteria usually emit bluish-green light with a peak emission wavelength (λ(max) ) at about 490?nm. Some species belonging to the genus Photobacterium are exceptions, producing an accessory blue fluorescent protein (lumazine protein: LumP) that causes a blue shift, from λ(max) ≈?490 to λ(max) ≈?476?nm. However, the incidence of blue-shifted light emission or the presence of accessory fluorescent proteins in bacteria of the genus Vibrio has never been reported. From our spectral analysis of light emitted by 16 luminous strains of the genus Vibrio, it was revealed that most strains of Vibrio azureus emit a blue-shifted light with a peak at approximately 472?nm, whereas other Vibrio strains emit light with a peak at around 482?nm. Therefore, we investigated the mechanism underlying this blue shift in V.?azureus NBRC 104587(T) . Here, we describe the blue-shifted light emission spectra and the isolation of a blue fluorescent protein. Intracellular protein analyses showed that this strain had a blue fluorescent protein (that we termed VA-BFP), the fluorescent spectrum of which was almost identical to that of the in vivo light emission spectrum of the strain. This result strongly suggested that VA-BFP was responsible for the blue-shifted light emission of V.?azureus.     

An alternative excited-state proton transfer pathway in green fluorescent protein variant S205V

Wild-type green fluorescent protein (wt-GFP) has a prominent absorbance band centered at approximately 395 nm, attributed to the neutral chromophore form. The green emission arising upon excitation of this band results from excited-state proton transfer (ESPT) from the chromophore hydroxyl, through a hydrogen-bond network proposed to consist of a water molecule and Ser205, to Glu222. Although evidence for Glu222 as a terminal proton acceptor has already been obtained, no evidence for the participation of Ser205 in the proton transfer process exists. To examine the role of Ser205 in the proton transfer, we mutated Ser205 to valine. However, the derived GFP variant S205V, upon excitation at 400 nm, still produces green fluorescence. Time-resolved emission spectroscopy suggests that ESPT contributes to the green fluorescence, and that the proton transfer takes place approximately 30 times more slowly than in wt-GFP. The crystal structure of S205V reveals rearrangement of Glu222 and Thr203, forming a new hydrogen-bonding network. We propose this network to be an alternative ESPT pathway with distinctive features that explain the significantly slowed rate of proton transfer. In support of this proposal, the double mutant S205V/T203V is shown to be a novel blue fluorescent protein containing a tyrosine-based chromophore, yet is incapable of ESPT. The results have implications for the detailed mechanism of ESPT and the photocycle of wt-GFP, in particular for the structures of spectroscopically identified intermediates in the cycle.     

Enhanced dynamic range in a genetically encoded Ca2+ sensor

Genetically encoded fluorescence resonance energy transfer (FRET) indicators are powerful tools for real-time detection of second messenger molecules and activation of signal proteins. However, these fluorescent protein-based sensors typically display marginal FRET efficiency. To improve their FRET efficiency for optical imaging and screening, we developed a number of fluorescent protein mutants based on cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). To improve FRET ratios, which were initially within a narrow dynamic range, we used DNA shuffling to develop a new FRET pair called 3xCFP/Venus. The optimized 3xCFP/Venus pair exhibited higher FRET ratios than CyPet/YPet, which has one of the greatest dynamic ranges of protein-based FRET pairs. We converted this FRET pair to a Ca(2+) FRET indicators using circular permutation Venus (cpVenus) linked with 3xCFP to form 3xCFP/cpVenus, which displayed an ～11-fold change in dynamic range in response to Ca(2+) binding. The enhanced dynamic range for Ca(2+) concentration detection using 3xCFP/cpVenus was confirmed in PC12 cells using previously established indicators (TN-XXL, ECFP/cpCitrine). To our knowledge, this FRET pair displays the largest dynamic range so far among genetically-encoded sensors, and can be used for sensitive FRET detection.     

The photophysics of green fluorescent protein: influence of the key amino acids at positions 65, 203, and 222

The three amino acids S65, T203, and E222 crucially determine the photophysical behavior of wild-type green fluorescent protein. We investigate the impact of four point mutations at these positions and their respective combinations on green fluorescent protein's photophysics using absorption spectroscopy, as well as steady-state and time-resolved fluorescence spectroscopy. Our results highlight the influence of the protein's hydrogen-bonding network on the equilibrium between the different chromophore states and on the efficiency of the excited-state proton transfer. The mutagenic approach allows us to separate different mechanisms responsible for fluorescence quenching, some of which were previously discussed theoretically. Our results will be useful for the development of new strategies for the generation of autofluorescent proteins with specific photophysical properties. One example presented here is a variant exhibiting uncommon blue fluorescence.     

Enhanced dynamic range in a genetically encoded Ca sensor

Genetically encoded fluorescence resonance energy transfer (FRET) indicators are powerful tools for real-time detection of second messenger molecules and activation of signal proteins. However, these fluorescent protein-based sensors typically display marginal FRET efficiency. To improve their FRET efficiency for optical imaging and screening, we developed a number of fluorescent protein mutants based on cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP). To improve FRET ratios, which were initially within a narrow dynamic range, we used DNA shuffling to develop a new FRET pair called 3xCFP/Venus. The optimized 3xCFP/Venus pair exhibited higher FRET ratios than CyPet/YPet, which has one of the greatest dynamic ranges of protein-based FRET pairs. We converted this FRET pair to a Ca²⁺ FRET indicators using circular permutation Venus (cpVenus) linked with 3xCFP to form 3xCFP/cpVenus, which displayed an ∼11-fold change in dynamic range in response to Ca²⁺ binding. The enhanced dynamic range for Ca²⁺ concentration detection using 3xCFP/cpVenus was confirmed in PC12 cells using previously established indicators (TN-XXL, ECFP/cpCitrine). To our knowledge, this FRET pair displays the largest dynamic range so far among genetically-encoded sensors, and can be used for sensitive FRET detection.     

Dual‐color‐emitting green fluorescent protein from the sea cactus Cavernularia obesa and its use as a pH indicator for fluorescence microscopy

We isolated and characterized a green fluorescent protein (GFP) from the sea cactus Cavernularia obesa. This GFP exists as a dimer and has absorption maxima at 388 and 498 nm. Excitation at 388 nm leads to blue fluorescence (456 nm maximum) at pH 5 and below, and green fluorescence (507 nm maximum) at pH 7 and above, and the GFP is remarkably stable at pH 4. Excitation at 498 nm leads to green fluorescence (507 nm maximum) from pH 5 to pH 9. We introduced five amino acid substitutions so that this GFP formed monomers rather than dimers and then used this monomeric form to visualize intracellular pH change during the phagocytosis of living cells by use of fluorescence microscopy. The intracellular pH change is visualized by use of a simple long‐pass emission filter with single‐wavelength excitation, which is technically easier to use than dual‐emission fluorescent proteins that require dual‐wavelength excitation. Copyright © 2013 John Wiley & Sons, Ltd.     

Interaction of a hydrophobic fluorescent probe with mouse embryo fibroblasts

Fluorescence photomicrographs show that the hydrophobic fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS) binds to hydrophobic components of intact 3T3 cells. Cells exposed to ANS exhibit fluorescence in the cytoplasm, intense nuclear membrane fluorescence, and well-defined fluorescent nucleoli. Fluorescence titrations of 3T3 cells with ANS show a decrease in fluorescence intensity, a blue shift of native cell emission with increasing ANS concentration and the appearance of a new peak due to ANS fluorescence. These fluorescence effects are ascribed to energy transfer processes involving bound ANS and the tryptophan and tyrosine residues of cellular proteins. ANS bound to 3T3 cells appears to quench the long wavelength component of the cellular tryptophan fluorescence, resulting in an unmasking of tryptophan and tyrosine emission at shorter wavelengths.     

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.     

Biophysical characterization of natural and mutant fluorescent proteins cloned from zooxanthellate corals

Two novel colored fluorescent proteins were cloned and biophysically characterized from zooxanthellate corals (Anthozoa). A cyan fluorescent protein derived from the coral Montastrea cavernosa (mcCFP) is a trimeric complex with strong blue-shifted excitation and emission maxima at 432 and 477 nm, respectively. The native complex has a fluorescence lifetime of 2.66 ± 0.01 ns and an inferred absolute quantum yield of 0.385. The spectroscopic properties of a green fluorescent protein cloned from Meandrina meandrites (mmGFP) resemble the commercially available GFP derived originally from the hydrozoan Aequorea victoria (avGFP). mmGFP is a monomeric protein with an excitation maximum at 398 nm and an emission maximum at 505 nm, a fluorescence lifetime of 3.10 ± 0.01 ns and an absolute quantum yield of 0.645. Sequence homology with avGFP and the red fluorescent protein (DsRed) indicates that the proteins adopt the classic β-barrel configuration with 11 β-strands. The three amino acid residues that comprise the chromophore are QYG for mcCFP and TYG for mmGFP, compared with SYG for avGFP. A single point mutation, Ser-110 to Asn, was introduced into mmGFP by random mutagenesis. Denaturation and refolding experiments showed that the mutant has reduced aggregation, increased solubility and more efficient refolding relative to the wild type. Time-resolved emission lifetimes and anisotropies suggest that the electronic structure of the chromophore is highly dependent on the protonation state of adjoining residues.     

Spectral and structural analysis of a red fluorescent protein from Acropora digitifera

Fluorescent proteins (FPs) possess a wide variety of spectral properties that make them of widespread interest as optical markers. These proteins can be applied as pH indicators or metal biosensors. The discovery and characterization of new fluorescent proteins is expected to further extend their application. Here, we report the spectral and structural analysis of a red fluorescent protein from Acropora digitifera (designated AdRed). This protein shows a tetrameric state and is red emitting, with excitation and emission maxima at 567 and 612 nm, respectively. Its crystal structure shows the tetrameric interface stabilized by hydrogen bonding and salt bridges. The electron density map of the chromophore, consisting of Asp66–Tyr67–Gly68, shows the decarboxylated side chain of Asp66. Ser223, located near the chromophore, has the role of bridging His202 and Glu221, and is part of the hydrogen bond network. Mutated AdRed with Cys148Ser reveals a blue shift in fluorescence excitation and emission. Our results provide insights into understanding the molecular function of AdRed and other FPs.     

Resonance energy transfer between green fluorescent protein variants: complexities revealed with myosin fusion proteins

Green fluorescent protein and its variants are frequently used as F?rster (fluorescence) resonance energy transfer (FRET) pairs to determine the proximity of protein domains. We prepared fusion proteins comprising yellow fluorescent protein-Dictyostelium myosin II motor domain-cyan fluorescent protein (YFP-myosin-CFP) and compared their FRET properties with an existing construct (GFP-myosin-BFP), containing a green fluorescent protein acceptor and blue fluorescent protein donor [Suzuki, Y., Yasunaga, T., Ohkura, R., Wakabayashi, T. and Sutoh, K. (1998) Nature 396, 380-383]. The latter construct showed an apparent 40% reduction in acceptor fluorescence on ATP addition, when excited via the donor, compared with the YFP-myosin-CFP constructs which showed a small increase (相似文献   

Ratiometric detection of Zn(II) using chelating fluorescent protein chimeras

Fluorescent indicators for the real-time imaging of small molecules or metal ions in living cells are invaluable tools for understanding their physiological function. Genetically encoded sensors based on fluorescence resonance energy transfer (FRET) between fluorescent protein domains have important advantages over synthetic probes, but often suffer from a small ratiometric change. Here, we present a new design approach to obtain sensors with a large difference in emission ratio between the bound and unbound states. De novo Zn(II)-binding sites were introduced directly at the surface of both fluorescent domains of a chimera of enhanced cyan and yellow fluorescent protein, connected by a flexible peptide linker. The resulting sensor ZinCh displayed an almost fourfold change in fluorescence emission ratio upon binding of Zn(II). Besides a high affinity for Zn(II), the sensor was shown to be selective over other physiologically relevant metal ions. Its unique biphasic Zn(II)-binding behavior could be attributed to the presence of two distinct Zn(II)-binding sites and allowed ratiometric fluorescent detection of Zn(II) over a concentration range from 10 nM to 1 mM. Size-exclusion chromatography and fluorescence anisotropy were used to provide a detailed picture of the conformational changes associated with each Zn(II)-binding step. The high affinity for Zn(II) was mainly due to a high effective concentration of the fluorescent proteins and could be understood quantitatively by modeling the peptide linker between the fluorescent proteins as a random coil. The strategy of using chelating fluorescent protein chimeras to develop FRET sensor proteins with a high ratiometric change is expected to be more generally applicable, in particular for other metal ions and small molecules.     

Engineered metal binding sites on green fluorescence protein

The ability to assay a variety of metals by noninvasive methods has applications in both biomedical and environmental research. Green fluorescent protein (GFP) is a protein isolated from coelenterates that exhibits spontaneous fluorescence. GFP does not require any exogenous cofactors for fluorescence, and can be easily appended to other proteins at the DNA level, producing a fluorescence-labeled target protein in vivo. Metals in close proximity to chromophores are known to quench fluorescence in a distance-dependent fashion. Potential metal binding sites on the surface of GFP have been identified and mutant proteins have been designed, created, and characterized. These metal-binding mutants of GFP exhibit fluorescence quenching at lower transition metal ion concentrations than those of the wild-type protein. These GFP mutants represent a new class of protein-based metal sensors.     

Spectrofluorimetric analysis of 7-hydroxycoumarin binding to bovine phenol sulfotransferase

Phenol sulfotransferases (SULT1s, EC 2.8.2.1) catalyze sulfuryl group transfer from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to the hydroxyl oxygen of aromatic acceptor substrates. Previous work with the bovine SULT1A1 has utilized the highly fluorescent substrate 7-hydroxycoumarin (7-HC, umbelliferone) as an acceptor substrate [Biochem. Biophys. Res. Commun. 261 (1999) 815]. Here we report that adenosine-3',5'-bisphosphate (PAP)-dependent binding of 7-HC to bSULT1A1 can be observed due to the appearance of a 400-420-nm shoulder in the emission spectrum, using an excitation wavelength of 280 nm. This emission was observed by placing 7-HC in ethanol, which is consistent with bSULT1A1 phenol binding site hydrophobicity. Titrations with 7-HC indicate a K(d) for 7-HC of 0.58 microM and substoichiometric binding to the homodimeric enzyme. The bSULT1A1:PAP:7-HC complex could be disrupted with pentachlorophenol (PCP), titrations with which indicated 0.5 equivalents per enzyme subunit. Titrations of enzyme plus 7-HC with PAP also indicated 0.5 equivalents per enzyme subunit. These results suggest a model of homodimeric bSULT1A1 in which subunit interactions favor half-site reactivity in the formation of a dead end complex.     

Detergent screening of the human voltage‐gated proton channel using fluorescence‐detection size‐exclusion chromatography

The human voltage‐gated proton channel (Hv1) is a membrane protein consisting of four transmembrane domains and intracellular amino‐ and carboxy‐termini. The protein is activated by membrane depolarization, similar to other voltage‐sensitive proteins. However, the Hv1 proton channel lacks a traditional ion pore. The human Hv1 proton channel has been implicated in mediating sperm capacitance, stroke, and most recently as a biomarker/mediator of cancer metastasis. Recently, the three‐dimensional structures for homologues of this voltage‐gated proton channel were reported. However, it is not clear what artificial environment is needed to facilitate the isolation and purification of the human Hv1 proton channel for structural study. In the present study, we generated a chimeric protein that placed an enhanced green fluorescent protein (EGFP) to the amino‐terminus of the human Hv1 proton channel (termed EGFP‐Hv1). The chimeric protein was expressed in a baculovirus expression system using Sf9 cells and subjected to detergent screening using fluorescence‐detection size‐exclusion chromatography. The EGFP‐Hv1 proton channel can be solubilized in the zwitterionic detergent Anzergent 3–12 and the nonionic n‐dodecyl‐β‐d ‐maltoside (DDM) with little protein aggregation and a prominent monomeric protein peak at 48 h postinfection. Furthermore, we demonstrate that the chimeric protein exhibits a monomeric protein peak, which is distinguishable from protein aggregates, at the final size‐exclusion chromatography purification step. Taken together, we can conclude that solubilization in DDM will provide a useable final product for further structural characterization of the full‐length human Hv1 proton channel.     

Studies of fluorescence inDrosophila compound eyes: changes induced by intense light and vitamin A deprivation

Summary Ultraviolet light excites a red fluorescence fromDrosophila R1–6 rhabdomeres which is superimposed on a blue background emission. Metarhodopsin (M570) pigment generates some or all of the vitamin A dependent red emission. However, the excitation spectrum for red emission peaks in the UV. This suggests that the pigment which sensitizes R1–6's visual pigment to UV light (sensitizing pigment) absorbs the UV light, sensitizing metarhodopsin's fluorescence by energy transfer. Blue emission is neither from sensitizing pigment nor from visual pigment as shown by vitamin A deprivation studies.Very intense UV or blue stimulation causes these changes: (1) conversion of visual pigment into a fluorescent product; (2) destruction of this fluorescent product; (3) a decrease in the blue background fluorescence (even in vitamin A deprived flies); and (4) a permanent destruction of visual pigment and retinal degeneration. The first effect requires intensities 3 log units brighter than needed to interconvert rhodopsin and metarhodopsin 1/2 way to photoequilibrium. UV light is about 5 times as effective as blue light for the conversion of visual pigment into fluorescent product.     

Synthesis and spectroscopic characterisation of new ESIPT fluorescent protein probes.

Three new benzazole isothiocyanate fluorescent dyes, 2-(4'-isothiocyanate-2'-hydroxyphenyl)benzoxazole, 2-(4'-isothiocyanate-2'-hydroxyphenyl)benzothiazole and 2-(4'-isothiocyanate-2'-hydroxyphenyl)benzimidazole were synthesised, purified until optical purity grade and characterised by spectroscopic techniques. UV/VIS and steady-state fluorescence were also applied to characterise the photophysical behaviour of the dyes. These dyes exhibit an intense fluorescence emission with a large Stokes shift, inherent to the class of benzazoles which relax by the excited state intramolecular proton transfer (ESIPT) mechanism. The dyes were studied for labeling bovine serum albumin (BSA), resulting conjugates BSA-dye with a remarkable photostability under UV/VIS radiation in relation to classical protein labels. The resulting conjugates presented fluorescence in the blue-green region. Direct fluorescence detection of protein-labeled with those dyes after polyacrylamide gel electrophoresis indicates their potential use as fluorescent probes for proteins.     

Exploration of new chromophore structures leads to the identification of improved blue fluorescent proteins

The variant of Aequorea green fluorescent protein (GFP) known as blue fluorescent protein (BFP) was originally engineered by substituting histidine for tyrosine in the chromophore precursor sequence. Herein we report improved versions of BFP along with a variety of engineered fluorescent protein variants with novel and distinct chromophore structures that all share the property of a blue fluorescent hue. The two most intriguing of the new variants are a version of GFP in which the chromophore does not undergo excited-state proton transfer and a version of mCherry with a phenylalanine-derived chromophore. All of the new blue fluorescing proteins have been critically assessed for their utility in live cell fluorescent imaging. These new variants should greatly facilitate multicolor fluorescent imaging by legitimizing blue fluorescing proteins as practical and robust members of the fluorescent protein "toolkit".