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 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 Mutations of β-arrestin 2 that limit self-association also interfere with interactions with the β2-adrenoceptor and the ERK1/2 MAPKs: implications for β2-adrenoceptor signalling via the ERK1/2 MAPKs

2008 ◽  
Vol 413 (1) ◽  
pp. 51-60 ◽  
Author(s):  
Tian-Rui Xu ◽  
George S. Baillie ◽  
Narinder Bhari ◽  
Thomas M. Houslay ◽  
Andrew M. Pitt ◽  
...  
Keyword(s):  
Amino Acids ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Limited Proteolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Peptide Arrays ◽  
Enhanced Yellow Fluorescent Protein ◽  
Self Association ◽  
Immobilized Peptide

FRET (fluorescence resonance energy transfer) and co-immunoprecipitation studies confirmed the capacity of β-arrestin 2 to self-associate. Amino acids potentially involved in direct protein–protein interaction were identified via combinations of spot-immobilized peptide arrays and mapping of surface exposure. Among potential key amino acids, Lys285, Arg286 and Lys295 are part of a continuous surface epitope located in the polar core between the N- and C-terminal domains. Introduction of K285A/R286A mutations into β-arrestin 2–eCFP (where eCFP is enhanced cyan fluorescent protein) and β-arrestin 2–eYFP (where eYFP is enhanced yellow fluorescent protein) constructs substantially reduced FRET, whereas introduction of a K295A mutation had a more limited effect. Neither of these mutants was able to promote β2-adrenoceptor-mediated phosphorylation of the ERK1/2 (extracellular-signal-regulated kinase 1/2) MAPKs (mitogen-activated protein kinases). Both β-arrestin 2 mutants displayed limited capacity to co-immunoprecipitate ERK1/2 and further spot-immobilized peptide arrays indicated each of Lys285, Arg286 and particularly Lys295 to be important for this interaction. Direct interactions between β-arrestin 2 and the β2-adrenoceptor were also compromised by both K285A/R286A and K295A mutations of β-arrestin 2. These were not non-specific effects linked to improper folding of β-arrestin 2 as limited proteolysis was unable to distinguish the K285A/R286A or K295A mutants from wild-type β-arrestin 2, and the interaction of β-arrestin 2 with JNK3 (c-Jun N-terminal kinase 3) was unaffected by the K285A/R286A or L295A mutations. These results suggest that amino acids important for self-association of β-arrestin 2 also play an important role in the interaction with both the β2-adrenoceptor and the ERK1/2 MAPKs. Regulation of β-arrestin 2 self-association may therefore control β-arrestin 2-mediated β2-adrenoceptor-ERK1/2 MAPK signalling.

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 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.

scholarly journals Enhancement of correct protein folding in vivo by a non-lytic baculovirus

2004 ◽  
Vol 382 (2) ◽  
pp. 695-702 ◽  
Author(s):  
Yu HO ◽  
Huei-Ru LO ◽  
Tzu-Ching LEE ◽  
Carol P. Y. WU ◽  
Yu-Chan CHAO
Keyword(s):  
Energy Transfer ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Random Mutagenesis ◽  
Resonance Energy ◽  
Vector System ◽  
Enhanced Yellow Fluorescent Protein

The BEVS (baculovirus expression vector system) is widely used for the production of proteins. However, engineered proteins frequently experience the problem of degradation, possibly due to the lytic nature of the conventional BEVS (herein referred to as L-BEVS). In the present study, a non-lytic BEVS (N-BEVS) was established by random mutagenesis of viral genomes. At 5 days post-infection, N-BEVS showed only 7% cell lysis, whereas L-BEVS showed 60% lysis of cells. The quality of protein expressed in both N- and L-BEVSs was examined further using a novel FRET (fluorescence resonance energy transfer)-based assay. To achieve this, we constructed a concatenated fusion protein comprising LUC (luciferase) sandwiched between EYFP (enhanced yellow fluorescent protein) and ECFP (enhanced cyan fluorescent protein). The distance separating the two fluorescent proteins in the fusion protein EYFP–LUC–ECFP (designated hereafter as the YLC construct) governs energy transfer between EYFP and ECFP. FRET efficiency thus reflects the compactness of LUC, indicating its folding status. We found more efficient FRET in N-BEVS compared with that obtained in L-BEVS, suggesting that more tightly folded LUC was produced in N-BEVS. YLC expression was also analysed by Western blotting, revealing significantly less protein degradation in N-BEVS than in L-BEVS, in which extensive degradation was observed. This FRET-based in vivo folding technology showed that YLC produced in N-BEVS is more compact, correlating with improved resistance to degradation. N-BEVS is thus a convenient alternative for L-BEVS for the production of proteins vulnerable to degradation using baculoviruses.

scholarly journals Fluorescent sensors for activity and regulation of the nitrate transceptor CHL1/NRT1.1 and oligopeptide transporters

2014 ◽  
Author(s):  
Cheng Hsun Ho ◽  
Wolf B. Frommer
Keyword(s):  
Fluorescent Protein ◽  
New Technology ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Peptide Transport ◽  
Transport Activity ◽  
Proton Coupling ◽  
Activity Sensors

To monitor nitrate and peptide transport activity in vivo, we converted the dual-affinity nitrate transceptor CHL1/NRT1.1/NPF6.3 and four related oligopeptide transporters PTR1, 2, 4 and 5 into fluorescence activity sensors (NiTrac1, PepTrac). Substrate addition to yeast expressing transporter fusions with yellow fluorescent protein and mCerulean triggered substrate-dependent donor quenching or resonance energy transfer. Fluorescence changes were nitrate/peptide-specific, respectively. Like CHL1, NiTrac1 had biphasic kinetics. Mutation of T101A eliminated high-affinity transport and blocked the fluorescence response to low nitrate. NiTrac was used for characterizing side chains considered important for substrate interaction, proton coupling, and regulation. We observed a striking correlation between transport activity and sensor output. Coexpression of NiTrac with known calcineurin-like proteins (CBL1, 9; CIPK23) and candidates identified in an interactome screen (CBL1, KT2, WNKinase 8) blocked NiTrac1 responses, demonstrating the suitability for in vivo analysis of activity and regulation. The new technology is applicable in plant and medical research.

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.

Sign in / Sign up

Export Citation Format

Share Document