We report a monomeric yellow-green fluorescent protein, mNeonGreen, derived from a tetrameric fluorescent protein from the cephalochordate Branchiostoma lanceolatum. mNeonGreen is the brightest ...monomeric green or yellow fluorescent protein yet described to our knowledge, performs exceptionally well as a fusion tag for traditional imaging as well as stochastic single-molecule superresolution imaging and is an excellent fluorescence resonance energy transfer (FRET) acceptor for the newest cyan fluorescent proteins.
Advances in fluorescent protein technology Shaner, Nathan C; Patterson, George H; Davidson, Michael W
Journal of cell science,
12/2007, Letnik:
120, Številka:
24
Journal Article
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Current fluorescent protein (FP) development strategies are focused on fine-tuning the photophysical properties of blue to yellow variants derived from the Aequorea victoria jellyfish green ...fluorescent protein (GFP) and on the development of monomeric FPs from other organisms that emit in the yellow-orange to far-red regions of the visible light spectrum. Progress toward these goals has been substantial, and near-infrared emitting FPs may loom over the horizon. The latest efforts in jellyfish variants have resulted in new and improved monomeric BFP, CFP, GFP and YFP variants, and the relentless search for a bright, monomeric and fast-maturing red FP has yielded a host of excellent candidates, although none is yet optimal for all applications. Meanwhile, photoactivatable FPs are emerging as a powerful class of probes for intracellular dynamics and, unexpectedly, as useful tools for the development of superresolution microscopy applications.
MCherry, the Discosoma sp. mushroom coral-derived monomeric red fluorescent protein (RFP), is a commonly used genetically encoded fluorophore for live cell fluorescence imaging. We have used a ...combination of protein design and directed evolution to develop mCherry variants with low cytotoxicity to Escherichia coli and altered excitation and emission profiles. These efforts ultimately led to a long Stokes shift (LSS)-mCherry variant (λex = 460 nm and λem = 610 nm) and a red-shifted (RDS)-mCherry variant (λex = 600 nm and λem = 630 nm). These new RFPs provide insight into the influence of the chromophore environment on mCherry's fluorescence properties, and may serve as templates for the future development of fluorescent probes for live cell imaging.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
All organic fluorophores undergo irreversible photobleaching during prolonged illumination. Although fluorescent proteins typically bleach at a substantially slower rate than many small-molecule ...dyes, in many cases the lack of sufficient photostability remains an important limiting factor for experiments requiring large numbers of images of single cells. Screening methods focusing solely on brightness or wavelength are highly effective in optimizing both properties, but the absence of selective pressure for photostability in such screens leads to unpredictable photobleaching behavior in the resulting fluorescent proteins. Here we describe an assay for screening libraries of fluorescent proteins for enhanced photostability. With this assay, we developed highly photostable variants of mOrange (a wavelength-shifted monomeric derivative of DsRed from Discosoma sp.) and TagRFP (a monomeric derivative of eqFP578 from Entacmaea quadricolor) that maintain most of the beneficial qualities of the original proteins and perform as reliably as Aequorea victoria GFP derivatives in fusion constructs.
The recent explosion in the diversity of available fluorescent proteins (FPs) promises a wide variety of new tools for biological imaging. With no unified standard for assessing these tools, however, ...a researcher is faced with difficult questions. Which FPs are best for general use? Which are the brightest? What additional factors determine which are best for a given experiment? Although in many cases, a trial-and-error approach may still be necessary in determining the answers to these questions, a unified characterization of the best available FPs provides a useful guide in narrowing down the options.
The performance of Förster Resonance Energy Transfer (FRET) biosensors depends on brightness and photostability, which are dependent on the characteristics of the fluorescent proteins that are ...employed. Yellow fluorescent protein (YFP) is often used as an acceptor but YFP is prone to photobleaching and pH changes. In this study, we evaluated the properties of a diverse set of acceptor fluorescent proteins in combination with the optimized CFP variant mTurquoise2 as the donor. To determine the theoretical performance of acceptors, the Förster radius was determined. The practical performance was determined by measuring FRET efficiency and photostability of tandem fusion proteins in mammalian cells. Our results show that mNeonGreen is the most efficient acceptor for mTurquoise2 and that the photostability is better than SYFP2. The non-fluorescent YFP variant sREACh is an efficient acceptor, which is useful in lifetime-based FRET experiments. Among the orange and red fluorescent proteins, mCherry and mScarlet-I are the best performing acceptors. Several new pairs were applied in a multimolecular FRET based sensor for detecting activation of a heterotrimeric G-protein by G-protein coupled receptors. Overall, the sensor with mNeonGreen as acceptor and mTurquoise2 as donor showed the highest dynamic range in ratiometric FRET imaging experiments with the G-protein sensor.
Using mRNA sequencing and de novo transcriptome assembly, we identified, cloned, and characterized 9 previously undiscovered fluorescent protein (FP) homologs from Aequorea victoria and a related ...Aequorea species, with most sequences highly divergent from A. victoria green fluorescent protein (avGFP). Among these FPs are the brightest green fluorescent protein (GFP) homolog yet characterized and a reversibly photochromic FP that responds to UV and blue light. Beyond green emitters, Aequorea species express purple- and blue-pigmented chromoproteins (CPs) with absorbances ranging from green to far-red, including 2 that are photoconvertible. X-ray crystallography revealed that Aequorea CPs contain a chemically novel chromophore with an unexpected crosslink to the main polypeptide chain. Because of the unique attributes of several of these newly discovered FPs, we expect that Aequorea will, once again, give rise to an entirely new generation of useful probes for bioimaging and biosensing.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Protein-based, fluorescent biosensors power basic research on cell signaling in health and disease, but their use in automated laboratories is limited. We have now created two live-cell assays, one ...for diacyl glycerol and another for cAMP, that are robust (Z′ > 0.7) and easily deployed on standard fluorescence plate readers. We describe the development of these assays, focusing on the parameters that were critical for optimization, in the hopes that the lessons learned can be generalized to the development of new biosensor-based assays.
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”.
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