scholarly journals Characterization of a spectrally diverse set of fluorescent proteins as FRET acceptors for mTurquoise2

2017 ◽  
Author(s):  
Marieke Mastop ◽  
Daphne S. Bindels ◽  
Nathan C. Shaner ◽  
Marten Postma ◽  
Theodorus W. J. Gadella ◽  
...  
Keyword(s):  
G Protein ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Dynamic Range ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Tandem Fusion ◽  
Red Fluorescent Proteins

AbstractGenetically encoded Förster Resonance Energy Transfer (FRET) based biosensors report on changes in biochemical states in single living cells. The performance of biosensors depends on their brightness and dynamic range, which are dependent on the characteristics of the fluorescent proteins that are employed. Cyan fluorescent protein (CFP) is frequently combined with yellow fluorescent protein (YFP) as FRET pair in biosensors. However, current YFPs are prone to photobleaching and pH changes. In addition, more efficient acceptors may yield biosensors that have higher contrast. 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, mChery 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. The sensor with mScarlet-I as acceptor and mTurquoise2 as donor shows a higher dynamic range in ratiometric FRET imaging experiments and less variability than with mCherry as acceptor, due to the high quantum yield and efficient maturation of mScarlet-I. 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.

scholarly journals Enhanced brightness of bacterial luciferase by bioluminescence resonance energy transfer

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tomomi Kaku ◽  
Kazunori Sugiura ◽  
Tetsuyuki Entani ◽  
Kenji Osabe ◽  
Takeharu Nagai
Keyword(s):  
Energy Transfer ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Color Change ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bioluminescence Resonance Energy Transfer ◽  
Bacterial Luciferase ◽  
Bacterial Bioluminescence

AbstractUsing the lux operon (luxCDABE) of bacterial bioluminescence system as an autonomous luminous reporter has been demonstrated in bacteria, plant and mammalian cells. However, applications of bacterial bioluminescence-based imaging have been limited because of its low brightness. Here, we engineered the bacterial luciferase (heterodimer of luxA and luxB) by fusion with Venus, a bright variant of yellow fluorescent protein, to induce bioluminescence resonance energy transfer (BRET). By using decanal as an externally added substrate, color change and ten-times enhancement of brightness was achieved in Escherichia coli when circularly permuted Venus was fused to the C-terminus of luxB. Expression of the Venus-fused luciferase in human embryonic kidney cell lines (HEK293T) or in Nicotiana benthamiana leaves together with the substrate biosynthesis-related genes (luxC, luxD and luxE) enhanced the autonomous bioluminescence. We believe the improved luciferase will forge the way towards the potential development of autobioluminescent reporter system allowing spatiotemporal imaging in live cells.

scholarly journals Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay

2019 ◽  
Vol 20 (16) ◽  
pp. 3859 ◽  
Author(s):  
Michael Winkler ◽  
Florian Wrensch ◽  
Pascale Bosch ◽  
Maike Knoth ◽  
Michael Schindler ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Protein Interactions ◽  
Viral Entry ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Protein Protein Interactions

The interferon-induced transmembrane proteins 1–3 (IFITM1–3) inhibit host cell entry of several viruses. However, it is incompletely understood how IFITM1–3 exert antiviral activity. Two phenylalanine residues, F75 and F78, within the intramembrane domain 1 (IM1) were previously shown to be required for IFITM3/IFITM3 interactions and for inhibition of viral entry, suggesting that IFITM/IFITM interactions might be pivotal to antiviral activity. Here, we employed a fluorescence resonance energy transfer (FRET) assay to analyze IFITM/IFITM interactions. For assay calibration, we equipped two cytosolic, non-interacting proteins, super yellow fluorescent protein (SYFP) and super cyan fluorescent protein (SCFP), with signals that target proteins to membrane rafts and also analyzed a SCFP-SYFP fusion protein. This strategy allowed us to discriminate background signals resulting from colocalization of proteins at membrane subdomains from signals elicited by protein–protein interactions. Coexpression of IFITM1–3 and IFITM5 fused to fluorescent proteins elicited strong FRET signals, and mutation of F75 and F78 in IFITM3 (mutant IFITM3-FF) abrogated antiviral activity, as expected, but did not alter cellular localization and FRET signals. Moreover, IFITM3-FF co-immunoprecipitated efficiently with wild type (wt) IFITM3, lending further support to the finding that lack of antiviral activity of IFITM3-FF was not due to altered membrane targeting or abrogated IFITM3-IFITM3 interactions. Collectively, we report an assay that allows quantifying IFITM/IFITM interactions. Moreover, we confirm residues F75 and F78 as critical for antiviral activity but also show that these residues are dispensable for IFITM3 membrane localization and IFITM3/IFITM3 interactions.

scholarly journals Designing a Compact, Low-Cost FRET Microscopy Platform for the Undergraduate Classroom

2020 ◽  
Vol 1 (2) ◽  
Author(s):  
John W. Rupel ◽  
Sophia M. Sdao ◽  
Kadina E. Johnston ◽  
Ethan T. Nethery ◽  
Kaitlyn A. Gabardi ◽  
...  
Keyword(s):  
Undergraduate Students ◽  
Fluorescent Protein ◽  
Dynamic Range ◽  
Low Cost ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Light Sources ◽  
Effective Manner ◽  
Primary Mouse

ABSTRACT Advances in fluorescent biosensors allow researchers to spatiotemporally monitor a diversity of biochemical reactions and secondary messengers. However, commercial microscopes for the specific application of Förster Resonance Energy Transfer (FRET) are prohibitively expensive to implement in the undergraduate classroom, owing primarily to the dynamic range required and need for ratiometric emission imaging. The purpose of this article is to provide a workflow to design a low-cost, FRET-enabled microscope and to equip the reader with sufficient knowledge to compare commercial light sources, optics, and cameras to modify the device for a specific application. We used this approach to construct a microscope that was assembled by undergraduate students with no prior microscopy experience that is suitable for most single-cell cyan and yellow fluorescent protein FRET applications. The utility of this design was demonstrated by measuring small metabolic oscillations by using a lactate FRET sensor expressed in primary mouse pancreatic islets, highlighting the biologically suitable signal-to-noise ratio and dynamic range of our compact microscope. The instructions in this article provide an effective teaching tool for undergraduate educators and students interested in implementing FRET in a cost-effective manner.

scholarly journals Distance-Dependent Fluorescence Resonance Energy Transfer Enhancement on Nanoporous Gold

Nanomaterials ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 2927
Author(s):  
Lianmin Cui ◽  
Ling Zhang ◽  
Heping Zeng
Keyword(s):  
Energy Transfer ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Nanoporous Gold ◽  
Maximum Enhancement ◽  
Characteristic Energy ◽  
Fluorescence Resonance

Fluorescence resonance energy transfers (FRET) between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) on nanoporous gold (NPG) are systematically investigated by controlling the distance between NPG and fluorescent proteins with polyelectrolyte multilayers. The FRET between CFP and YFP is significantly enhanced by NPG, and the maximum enhancement is related to both ligament size of NPG and the distance between NPG and proteins. With the optimized distance, 18-fold FRET enhancement was obtained on NPG compared to that on glass, and the conversion efficiency is about 90%. The potential to tune the characteristic energy transfer distance has implications for applications in nanophotonic devices and provides a possible way to design sensors and light energy converters.

scholarly journals A splice variant of the G protein β3-subunit implicated in disease states does not modulate ion channels

2003 ◽  
Vol 13 (2) ◽  
pp. 85-95 ◽  
Author(s):  
Victor Ruiz-Velasco ◽  
Stephen R. Ikeda
Keyword(s):  
G Protein ◽  
Splice Variant ◽  
Fluorescent Protein ◽  
Sympathetic Neurons ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Protein Product ◽  
Enhanced Yellow Fluorescent Protein ◽  
Disease States

A single-nucleotide polymorphism (C825T) in the GNB3 gene produces an alternative splice variant of the heterotrimeric G protein β3 subunit (Gβ3). Translation of the alternatively spliced mRNA results in a protein product, Gβ3-s, in which 41 amino acids are deleted from Gβ3. Interestingly, previous studies indicate that the C825T allele occurs with a high frequency in patients with certain vascular disorders. However, little information is available regarding the functional role Gβ3-s might play in ion channel modulation. To examine this aspect, Gβ3 or Gβ3-s, along with either Gγ2 or Gγ5, were expressed in rat sympathetic neurons by nuclear microinjection of vector encoding the desired protein. In contrast to Gβ3, expression of Gβ3-s did not modulate N-type Ca2+ or G protein-gated inwardly rectifying K+ channels. In addition, Gβ3-s did not appear to complex with a pertussis toxin-insensitive mutant of Gαi2 or couple to natively expressed α2-adrenergic receptors. Finally, fluorescence resonance energy transfer (FRET) measurements indicated that enhanced yellow fluorescent protein (EYFP)-labeled Gβ3-s does not form a Gβγ heterodimer when coexpressed with enhanced cyan fluorescent protein (ECFP)-labeled Gγ2. Therefore, when expressed in sympathetic neurons, Gβ3-s appears to lack biological activity-hence pathological conditions in patients carrying the homozygous C825T allele may result from a functional knockout of Gβ3.

scholarly journals Single-Molecule Studies on a FRET Biosensor: Lessons from a Comparison of Fluorescent Protein Equipped versus Dye-Labeled Species

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3105 ◽  
Author(s):  
Henning Höfig ◽  
Michele Cerminara ◽  
Ilona Ritter ◽  
Antonie Schöne ◽  
Martina Pohl ◽  
...  
Keyword(s):  
Conformational Change ◽  
Single Molecule ◽  
Fluorescent Dyes ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Strong Increase ◽  
Single Molecule Level ◽  
Labeling Scheme

Bacterial periplasmic binding proteins (PBPs) undergo a pronounced ligand-induced conformational change which can be employed to monitor ligand concentrations. The most common strategy to take advantage of this conformational change for a biosensor design is to use a Förster resonance energy transfer (FRET) signal. This can be achieved by attaching either two fluorescent proteins (FPs) or two organic fluorescent dyes of different colors to the PBPs in order to obtain an optical readout signal which is closely related to the ligand concentration. In this study we compare a FP-equipped and a dye-labeled version of the glucose/galactose binding protein MglB at the single-molecule level. The comparison demonstrates that changes in the FRET signal upon glucose binding are more pronounced for the FP-equipped sensor construct as compared to the dye-labeled analog. Moreover, the FP-equipped sensor showed a strong increase of the FRET signal under crowding conditions whereas the dye-labeled sensor was not influenced by crowding. The choice of a labeling scheme should therefore be made depending on the application of a FRET-based sensor.

scholarly journals tdLanYFP, a yellow, bright, photostable and pH insensitive fluorescent protein for live cell imaging and FRET-based sensing strategies

2021 ◽  
Author(s):  
Y. Bousmah ◽  
H. Valenta ◽  
G. Bertolin ◽  
U. Singh ◽  
V. Nicolas ◽  
...  
Keyword(s):  
Single Molecule ◽  
Live Cell Imaging ◽  
Cell Imaging ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy ◽  
Live Cell ◽  
Live Cells

AbstractYellow fluorescent proteins (YFP) are widely used as optical reporters in Förster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. In fact, today, there is no yellow variant derived from the EYFP with a pK1/2 below ∼5.5. Here, we characterize a new yellow fluorescent protein, tdLanYFP, derived from the tetrameric protein from the cephalochordate B. lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133 000 mol−1.L.cm−1, it is, to our knowledge, the brightest dimeric fluorescent protein available, and brighter than most of the monomeric YFPs. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and preserves this property in live cells. As a consequence, tdLanYFP allows the imaging of cellular structures with sub-diffraction resolution with STED nanoscopy. We also demonstrate that the combination of high brightness and strong photostability is compatible with the use of spectro-microscopies in single molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pHs. Finally, we show that tdLanYFP can be a FRET partner either as donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFPa very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging that is also suitable for FRET experiment including at acidic pH.

scholarly journals Genetically Encoded Fluorescent Sensor for Poly-ADP-Ribose

2020 ◽  
Vol 21 (14) ◽  
pp. 5004
Author(s):  
Ekaterina O. Serebrovskaya ◽  
Nadezda M. Podvalnaya ◽  
Varvara V. Dudenkova ◽  
Anna S. Efremova ◽  
Nadya G. Gurskaya ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
Fluorescent Proteins ◽  
Fluorescent Sensor ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Post Translational Modification ◽  
Cellular Processes ◽  
Hydrogen Peroxide Treatment ◽  
Damage Response

Poly-(ADP-ribosyl)-ation (PARylation) is a reversible post-translational modification of proteins and DNA that plays an important role in various cellular processes such as DNA damage response, replication, transcription, and cell death. Here we designed a fully genetically encoded fluorescent sensor for poly-(ADP-ribose) (PAR) based on Förster resonance energy transfer (FRET). The WWE domain, which recognizes iso-ADP-ribose internal PAR-specific structural unit, was used as a PAR-targeting module. The sensor consisted of cyan Turquoise2 and yellow Venus fluorescent proteins, each in fusion with the WWE domain of RNF146 E3 ubiquitin ligase protein. This bipartite sensor named sPARroW (sensor for PAR relying on WWE) enabled monitoring of PAR accumulation and depletion in live mammalian cells in response to different stimuli, namely hydrogen peroxide treatment, UV irradiation and hyperthermia.

scholarly journals The luminescent HiBiT peptide enables selective quantitation of G protein–coupled receptor ligand engagement and internalization in living cells

2020 ◽  
Vol 295 (15) ◽  
pp. 5124-5135 ◽  
Author(s):  
Michelle E. Boursier ◽  
Sergiy Levin ◽  
Kris Zimmerman ◽  
Thomas Machleidt ◽  
Robin Hurst ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Cell Surface ◽  
G Protein ◽  
Dynamic Range ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Internalization ◽  
Cell Surface Receptor ◽  
Cellular Interactions ◽  
G Protein Coupled

G protein–coupled receptors (GPCRs) are prominent targets to new therapeutics for a range of diseases. Comprehensive assessments of their cellular interactions with bioactive compounds, particularly in a kinetic format, are imperative to the development of drugs with improved efficacy. Hence, we developed complementary cellular assays that enable equilibrium and real-time analyses of GPCR ligand engagement and consequent activation, measured as receptor internalization. These assays utilize GPCRs genetically fused to an N-terminal HiBiT peptide (1.3 kDa), which produces bright luminescence upon high-affinity complementation with LgBiT, an 18-kDa subunit derived from NanoLuc. The cell impermeability of LgBiT limits signal detection to the cell surface and enables measurements of ligand-induced internalization through changes in cell-surface receptor density. In addition, bioluminescent resonance energy transfer is used to quantify dynamic interactions between ligands and their cognate HiBiT-tagged GPCRs through competitive binding with fluorescent tracers. The sensitivity and dynamic range of these assays benefit from the specificity of bioluminescent resonance energy transfer and the high signal intensity of HiBiT/LgBiT without background luminescence from receptors present in intracellular compartments. These features allow analyses of challenging interactions having low selectivity or affinity and enable studies using endogenously tagged receptors. Using the β-adrenergic receptor family as a model, we demonstrate the versatility of these assays by utilizing the same HiBiT construct in analyses of multiple aspects of GPCR pharmacology. We anticipate that this combination of target engagement and proximal functional readout will prove useful to the study of other GPCR families and the development of new therapeutics.

scholarly journals Ultra-Sensitive Hydrogen Peroxide Sensor Based on Peroxiredoxin and Fluorescence Resonance Energy Transfer

2020 ◽  
Vol 10 (10) ◽  
pp. 3508
Author(s):  
Haijun Yu ◽  
Haoxiang Li ◽  
Yao Zhou ◽  
Shengmin Zhou ◽  
Ping Wang
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Disulfide Bonds ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Mixed Disulfide ◽  
Fluorescence Resonance ◽  
Protein Sensor

In this paper, a fluorescence resonance energy transfer (FRET)-based sensor for ultra-sensitive detection of H2O2 was developed by utilizing the unique enzymatic properties of peroxiredoxin (Prx) to H2O2. Cyan and yellow fluorescent protein (CFP and YFP) were fused to Prx and mutant thioredoxin (mTrx), respectively. In the presence of H2O2, Prx was oxidized into covalent homodimer through disulfide bonds, which were further reduced by mTrx to form a stable mixed disulfide bond intermediate between CFP-Prx and mTrx-YFP, inducing FRET. A linear quantification range of 10–320 nM was obtained according to the applied protein concentrations and the detection limit (LOD) was determined to be as low as 4 nM. By the assistance of glucose oxidase to transform glucose into H2O2, the CFP-Prx/mTrx-YFP system (CPmTY) was further exploited for the detection of glucose in real sample with good performance, suggesting this CPmTY protein sensor is highly practical.

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