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 The effect of proximity on the function and energy transfer capability of fluorescent protein pairs

2019 ◽  
Author(s):  
Jacob R. Pope ◽  
Rachel L. Johnson ◽  
W. David Jamieson ◽  
Harley L Worthy ◽  
Senthilkumar D. Kailasam ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Protein Protein Interactions ◽  
Fluorescent Properties ◽  
Transition Dipole ◽  
Green Fluorescent

AbstractFluorescent proteins (FPs) are commonly used in pairs to monitor dynamic biomolecular events through changes in their proximity via distance dependent processes such as Förster resonance energy transfer (FRET). Many FPs have a tendency to oligomerise, which is likely to be promoted through attachment to associating proteins through increases in local FP concentration. We show here that on association of FP pairs, the inherent function of the FPs can alter. Artificial dimers were constructed using a bioorthogonal Click chemistry approach that combined a commonly used green fluorescent protein (superfolder GFP) with itself, a yellow FP (Venus) or a red FP (mCherry). In each case dimerisation changes the inherent fluorescent properties, including FRET capability. The GFP homodimer demonstrated synergistic behaviour with the dimer being brighter than the sum of the two monomers. The structure of the GFP homodimer revealed that a water-rich interface is formed between the two monomers, with the chromophores being in close proximity with favourable transition dipole alignments. Dimerisation of GFP with Venus results in a complex displaying ∼86% FRET efficiency, which is significantly below the near 100% efficiency predicted. When GFP is complexed with mCherry, FRET and mCherry fluorescence itself is essentially lost. Thus, the simple assumptions used when monitoring interactions between proteins via FP FRET may not always hold true, especially under conditions whereby the protein-protein interactions promote FP interaction.Abstract Figure

scholarly journals Measuring ligand-dependent and ligand-independent interactions between nuclear receptors and associated proteins using Bioluminescence Resonance Energy Transfer (BRET2)

2006 ◽  
Vol 4 (1) ◽  
pp. nrs.04021 ◽  
Author(s):  
Kristen L. Koterba ◽  
Brian G. Rowan
Keyword(s):  
Energy Transfer ◽  
Protein Interactions ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Renilla Luciferase ◽  
Receptor Protein ◽  
Protein Protein Interactions

Bioluminescent resonance energy transfer (BRET2) is a recently developed technology for the measurement of protein-protein interactions in a live, cell-based system. BRET2 is characterized by the efficient transfer of excited energy between a bioluminescent donor molecule (Renilla luciferase) and a fluorescent acceptor molecule (a mutant of Green Fluorescent Protein (GFP2)). The BRET2 assay offers advantages over fluorescence resonance energy transfer (FRET) because it does not require an external light source thereby eliminating problems of photobleaching and autoflourescence. The absence of contamination by light results in low background that permits detection of very small changes in the BRET2 signal. BRET2 is dependent on the orientation and distance between two fusion proteins and therefore requires extensive preliminary standardization experiments to conclude a positive BRET2 signal independent of variations in protein titrations and arrangement in tertiary structures. Estrogen receptor (ER) signaling is modulated by steroid receptor coactivator 1 (SRC-1). To establish BRET2 in a ligand inducible system we used SRC-1 as the donor moiety and ER as the acceptor moiety. Expression and functionality of the fusion proteins were assessed by transient transfection in HEK-293 cells followed by Western blot analysis and measurement of ER-dependent reporter gene activity. These preliminary determinations are required prior to measuring nuclear receptor protein-protein interactions by BRET2. This article describes in detail the BRET2 methodology for measuring interaction between full-length ER and coregulator proteins in real-time, in an in vivo environment.

scholarly journals Dynamic tuning of FRET in a green fluorescent protein biosensor

2019 ◽  
Vol 5 (8) ◽  
pp. eaaw4988 ◽  
Author(s):  
Pablo Trigo-Mourino ◽  
Thomas Thestrup ◽  
Oliver Griesbeck ◽  
Christian Griesinger ◽  
Stefan Becker
Keyword(s):  
Green Fluorescent Protein ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Structural Data ◽  
X Ray ◽  
Dynamic Tuning ◽  
Green Fluorescent

Förster resonance energy transfer (FRET) between mutants of green fluorescent protein is widely used to monitor protein-protein interactions and as a readout mode in fluorescent biosensors. Despite the fundamental importance of distance and molecular angles of fluorophores to each other, structural details on fluorescent protein FRET have been missing. Here, we report the high-resolution x-ray structure of the fluorescent proteins mCerulean3 and cpVenus within the biosensor Twitch-2B, as they undergo FRET and characterize the dynamics of this biosensor with B02-dependent paramagnetic nuclear magnetic resonance at 900 MHz and 1.1 GHz. These structural data provide the unprecedented opportunity to calculate FRET from the x-ray structure and to compare it to experimental data in solution. We find that interdomain dynamics limits the FRET effect and show that a rigidification of the sensor further enhances FRET.

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 Oligomerization of the UDP-glucuronosyltransferase 1A Proteins

2006 ◽  
Vol 282 (7) ◽  
pp. 4821-4829 ◽  
Author(s):  
Theresa N. Operaña ◽  
Robert H. Tukey
Keyword(s):  
Protein Interactions ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Conclusive Evidence ◽  
Live Cells ◽  
Protein Protein Interactions ◽  
Dietary Nutrients ◽  
Membrane Bound

UDP-glucuronosyltransferases (UGTs) are membrane-bound proteins localized to the endoplasmic reticulum and catalyze the formation of β-d-glucopyranosiduronic acids (glucuronides) using UDP-glucuronic acid and acceptor substrates such as drugs, steroids, bile acids, xenobiotics, and dietary nutrients. Recent biochemical evidence indicates that the UGT proteins may oligomerize in the membrane, but conclusive evidence is still lacking. In the present study, we have used fluorescence resonance energy transfer (FRET) to study UGT1A oligomerization in live cells. This technique demonstrated that UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10 self-oligomerize (homodimerize). Heterodimer interactions were also explored, and it was determined that UGT1A1 was capable of binding with UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, and UGT1A10. In addition to the in vivo FRET analysis, UGT1A protein-protein interactions were demonstrated through co-immunoprecipitation experiments. Co-expression of hemagglutinin-tagged and cyan fluorescent protein-tagged UGT1A proteins, followed by immunoprecipitation with anti-hemagglutinin beads, illustrated the potential of each UGT1A protein to homodimerize. Co-immunoprecipitation results also confirmed that UGT1A1 was capable of forming heterodimer complexes with all of the UGT1A proteins, corroborating the FRET results in live cells. These preliminary studies suggest that the UGT1A family of proteins form oligomerized complexes in the membrane, a property that may influence function and substrate selectivity.

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 Heterodimerization Between the Lutropin and Follitropin Receptors is Associated With an Attenuation of Hormone-Dependent Signaling

Endocrinology ◽  
2013 ◽  
Vol 154 (10) ◽  
pp. 3925-3930 ◽  
Author(s):  
Xiuyan Feng ◽  
Meilin Zhang ◽  
Rongbin Guan ◽  
Deborah L. Segaloff
Keyword(s):  
Protein Interactions ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
G Protein Coupled Receptors ◽  
Fsh Receptor ◽  
Bioluminescence Resonance Energy Transfer ◽  
Protein Protein Interactions ◽  
Lh Receptor ◽  
G Protein Coupled ◽  
Stimulation Of

The LH receptor (LHR) and FSH receptor (FSHR) are each G protein-coupled receptors that play critical roles in reproductive endocrinology. Each of these receptors has previously been shown to self-associate into homodimers and oligomers shortly after their biosynthesis. As shown herein using bioluminescence resonance energy transfer to detect protein-protein interactions, our data show that the LHR and FSHR, when coexpressed in the same cells, specifically heterodimerize with each other. Further experiments confirm that at least a portion of the cellular LHR/FSHR heterodimers are present on the cell surface and are functional. We then sought to ascertain what effects, if any, heterodimerization between the LHR and FSHR might have on signaling. It was observed that when the LHR was expressed under conditions promoting the heterodimerization with FSHR, LH or human chorionic gonadotropin (hCG) stimulation of Gs was attenuated. Conversely, when the FSHR was expressed under conditions promoting heterodimerization with the LHR, FSH-stimulated Gs activation was attenuated. These results demonstrate that the coexpression of the LHR and FSHR enables heterodimerizaton between the 2 gonadotropin receptors and results in an attenuation of signaling through each receptor.

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