scholarly journals Resonance energy transfer imaging of phospholipid vesicle interaction with a planar phospholipid membrane: undulations and attachment sites in the region of calcium-mediated membrane--membrane adhesion.

1996 ◽  
Vol 107 (3) ◽  
pp. 329-351 ◽  
Author(s):  
W D Niles ◽  
J R Silvius ◽  
F S Cohen
Keyword(s):  
Energy Transfer ◽  
Membrane Fusion ◽  
Contact Region ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Phospholipid Vesicle ◽  
Osmotic Gradient ◽  
Attachment Sites ◽  
Donor And Acceptor ◽  
Planar Membrane

Membrane fusion of a phospholipid vesicle with a planar lipid bilayer is preceded by an initial prefusion stage in which a region of the vesicle membrane adheres to the planar membrane. A resonance energy transfer (RET) imaging microscope, with measured spectral transfer functions and a pair of radiometrically calibrated video cameras, was used to determine both the area of the contact region and the distances between the membranes within this zone. Large vesicles (5-20 microns diam) were labeled with the donor fluorophore coumarin-phosphatidylethanolamine (PE), while the planar membrane was labeled with the acceptor rhodamine-PE. The donor was excited with 390 nm light, and separate images of donor and acceptor emission were formed by the microscope. Distances between the membranes at each location in the image were determined from the RET rate constant (kt) computed from the acceptor:donor emission intensity ratio. In the absence of an osmotic gradient, the vesicles stably adhered to the planar membrane, and the dyes did not migrate between membranes. The region of contact was detected as an area of planar membrane, coincident with the vesicle image, over which rhodamine fluorescence was sensitized by RET. The total area of the contact region depended biphasically on the Ca2+ concentration, but the distance between the bilayers in this zone decreased with increasing [Ca2+]. The changes in area and separation were probably related to divalent cation effects on electrostatic screening and binding to charged membranes. At each [Ca2+], the intermembrane separation varied between 1 and 6 nm within each contact region, indicating membrane undulation prior to adhesion. Intermembrane separation distances < or = 2 nm were localized to discrete sites that formed in an ordered arrangement throughout the contact region. The area of the contact region occupied by these punctate attachment sites was increased at high [Ca2+]. Membrane fusion may be initiated at these sites of closest membrane apposition.

scholarly journals Distribution of a Glycosylphosphatidylinositol-anchored Protein at the Apical Surface of MDCK Cells Examined at a Resolution of <100 Å Using Imaging Fluorescence Resonance Energy Transfer

1998 ◽  
Vol 142 (1) ◽  
pp. 69-84 ◽  
Author(s):  
A.K. Kenworthy ◽  
M. Edidin
Keyword(s):  
Energy Transfer ◽  
Lipid Rafts ◽  
Fluorescence Resonance Energy Transfer ◽  
Mdck Cells ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Membrane Microdomains ◽  
Apical Surface ◽  
Donor And Acceptor ◽  
Fluorescence Resonance

Membrane microdomains (“lipid rafts”) enriched in glycosylphosphatidylinositol (GPI)-anchored proteins, glycosphingolipids, and cholesterol have been implicated in events ranging from membrane trafficking to signal transduction. Although there is biochemical evidence for such membrane microdomains, they have not been visualized by light or electron microscopy. To probe for microdomains enriched in GPI- anchored proteins in intact cell membranes, we used a novel form of digital microscopy, imaging fluorescence resonance energy transfer (FRET), which extends the resolution of fluorescence microscopy to the molecular level (&lt;100 Å). We detected significant energy transfer between donor- and acceptor-labeled antibodies against the GPI-anchored protein 5′ nucleotidase (5′ NT) at the apical membrane of MDCK cells. The efficiency of energy transfer correlated strongly with the surface density of the acceptor-labeled antibody. The FRET data conformed to theoretical predictions for two-dimensional FRET between randomly distributed molecules and were inconsistent with a model in which 5′ NT is constitutively clustered. Though we cannot completely exclude the possibility that some 5′ NT is in clusters, the data imply that most 5′ NT molecules are randomly distributed across the apical surface of MDCK cells. These findings constrain current models for lipid rafts and the membrane organization of GPI-anchored proteins.

Effect of compartmentalization of donor and acceptor on the ultrafast resonance energy transfer from DAPI to silver nanoclusters

Nanoscale ◽  
2016 ◽  
Vol 8 (26) ◽  
pp. 13006-13016 ◽  
Author(s):  
Roopali Prajapati ◽  
Surajit Chatterjee ◽  
Krishna K. Kannaujiya ◽  
Tushar Kanti Mukherjee
Keyword(s):  
Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Silver Nanoclusters ◽  
Donor And Acceptor

scholarly journals Versatility of Homogeneous Time-Resolved Fluorescence Resonance Energy Transfer Assays for Biologics Drug Discovery

2014 ◽  
Vol 20 (4) ◽  
pp. 508-518 ◽  
Author(s):  
Christine J. Rossant ◽  
Carl Matthews ◽  
Frances Neal ◽  
Caroline Colley ◽  
Matthew J. Gardener ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Drug Discovery ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved Fluorescence ◽  
Time Resolved ◽  
Donor And Acceptor ◽  
Wide Range ◽  
Fluorescence Resonance

Identification of potential lead antibodies in the drug discovery process requires the use of assays that not only measure binding of the antibody to the target molecule but assess a wide range of other characteristics. These include affinity ranking, measurement of their ability to inhibit relevant protein-protein interactions, assessment of their selectivity for the target protein, and determination of their species cross-reactivity profiles to support in vivo studies. Time-resolved fluorescence resonance energy transfer is a technology that offers the flexibility for development of such assays, through the availability of donor and acceptor fluorophore-conjugated reagents for detection of multiple tags or fusion proteins. The time-resolved component of the technology reduces potential assay interference, allowing screening of a range of different crude sample types derived from the bacterial or mammalian cell expression systems often used for antibody discovery projects. Here we describe the successful application of this technology across multiple projects targeting soluble proteins and demonstrate how it has provided key information for the isolation of potential therapeutic antibodies with the desired activity profile.

Measurement of proteases using chemiluminescence-resonance-energy-transfer chimaeras between green fluorescent protein and aequorin

2001 ◽  
Vol 357 (3) ◽  
pp. 687-697 ◽  
Author(s):  
Jonathan P. WAUD ◽  
Alexandra BERMÚDEZ FAJARDO ◽  
Thankiah SUDHAHARAN ◽  
Andrew R. TRIMBY ◽  
Jinny JEFFERY ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Green Fluorescent Protein ◽  
Caspase 3 ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dependent Manner ◽  
Cell Extracts ◽  
Donor And Acceptor ◽  
Green Fluorescent

Homogeneous assays, without a separation step, are essential for measuring chemical events in live cells and for drug discovery screens, and are desirable for making measurements in cell extracts or clinical samples. Here we demonstrate the principle of chemiluminescence resonance energy transfer (CRET) as a homogeneous assay system, using two proteases as models, one extracellular (α-thrombin) and the other intracellular (caspase-3). Chimaeras were engineered with aequorin as the chemiluminescent energy donor and green fluorescent protein (GFP) or enhanced GFP as the energy acceptors, with a protease linker (6 or 18 amino acid residues) recognition site between the donor and acceptor. Flash chemiluminescent spectra (20–60 s) showed that the spectra of chimaeras matched GFP, being similar to that of luminous jellyfish, justifying their designation as ‘Rainbow’ proteins. Addition of the protease shifted the emission spectrum to that of aequorin in a time- and dose-dependent manner. Separation of the proteolysed fragments showed that the ratio of green to blue light matched the extent of proteolysis. The caspase-3 Rainbow protein was able to provide information on the specificity of caspases in vitro and in vivo. It was also able to monitor caspase-3 activation in cells provoked into apoptosis by staurosporine (1 or 2μM). CRET can also monitor GFP fluor formation. The signal-to-noise ratio of our Rainbow proteins is superior to that of fluorescence resonance energy transfer, providing a potential platform for measuring agents that interact with the reactive site between the donor and acceptor.

scholarly journals Cyan-emitting and orange-emitting fluorescent proteins as a donor/acceptor pair for fluorescence resonance energy transfer

2004 ◽  
Vol 381 (1) ◽  
pp. 307-312 ◽  
Author(s):  
Satoshi KARASAWA ◽  
Toshio ARAKI ◽  
Takeharu NAGAI ◽  
Hideaki MIZUNO ◽  
Atsushi MIYAWAKI
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Fluorescent Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Acceptor Pair ◽  
Donor And Acceptor ◽  
Donor Acceptor ◽  
Donor Acceptor Pair ◽  
Fluorescence Resonance

GFP (green fluorescent protein)-based FRET (fluorescence resonance energy transfer) technology has facilitated the exploration of the spatio-temporal patterns of cellular signalling. While most studies have used cyan- and yellow-emitting FPs (fluorescent proteins) as FRET donors and acceptors respectively, this pair of proteins suffers from problems of pH-sensitivity and bleeding between channels. In the present paper, we demonstrate the use of an alternative additional donor/acceptor pair. We have cloned two genes encoding FPs from stony corals. We isolated a cyan-emitting FP from Acropara sp., whose tentacles exhibit cyan coloration. Similar to GFP from Renilla reniformis, the cyan FP forms a tight dimeric complex. We also discovered an orange-emitting FP from Fungia concinna. As the orange FP exists in a complex oligomeric structure, we converted this protein into a monomeric form through the introduction of three amino acid substitutions, recently reported to be effective for converting DsRed into a monomer (Clontech). We used the cyan FP and monomeric orange FP as a donor/acceptor pair to monitor the activity of caspase 3 during apoptosis. Due to the close spectral overlap of the donor emission and acceptor absorption (a large Förster distance), substantial pH-resistance of the donor fluorescence quantum yield and the acceptor absorbance, as well as good separation of the donor and acceptor signals, the new pair can be used for more effective quantitative FRET imaging.

scholarly journals BRED: bioluminescence energy transfer to dye for monitoring ceramide trafficking in cell

2021 ◽  
Author(s):  
Gita Naseri ◽  
Christoph Arenz
Keyword(s):  
Energy Transfer ◽  
High Efficiency ◽  
Single Cell Analysis ◽  
Specific Interaction ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cell Analysis ◽  
Bioluminescence Resonance Energy Transfer ◽  
Donor And Acceptor ◽  
Ligand Interactions

Bioluminescence resonance energy transfer (BRET) is a genetically encoded proximity-based tool to study biomolecular interactions. However, conventional BRET is usually restricted to only a few types of interactions like protein-protein or protein-ligand interactions. We here developed a spatially unbiased resonance energy transfer system, so-called BRED - bioluminescence resonance energy transfer to dye. BRED allows transferring energy from a genetically encoded bright human optimized luciferase to a fluorophore-labelled small molecule. The high efficiency of the system allows RET without specific interaction of donor and acceptor. Here, we applied BRED to monitor the trafficking of the signalling lipid ceramide, to the Golgi. This was enabled by an engineered Golgi-resident luciferase, which was used to sense the influx of BODIPY-labeled ceramide into the surrounding membrane. We demonstrated the implementation of the method via flow cytometry, thereby combining the sensitivity of bulk cell methods with the advantages of single-cell analysis. This toolbox enables simple and robust live-cell analysis of inhibitors of CERT-mediated ceramide transport. The design principle of our optogenetic tool can be applied to study intracellular trafficking of metabolites and screen for inhibitors of their key enzymes.

scholarly journals Two Orders of Magnitude Variation of Diffusion-Enhanced Förster Resonance Energy Transfer in Polypeptide Chains

Polymers ◽  
2018 ◽  
Vol 10 (10) ◽  
pp. 1079 ◽  
Author(s):  
Maik Jacob ◽  
Indrajit Ghosh ◽  
Roy D’Souza ◽  
Werner Nau
Keyword(s):  
Energy Transfer ◽  
Dynamic Properties ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Forster Resonance Energy Transfer ◽  
Förster Resonance Energy Transfer ◽  
Donor And Acceptor ◽  
The Impact ◽  
And Diffusion ◽  
Förster Resonance

A flexible peptide chain displays structural and dynamic properties that correspond to its folding and biological activity. These properties are mirrored in intrachain site-to-site distances and diffusion coefficients of mutual site-to-site motion. Both distance distribution and diffusion determine the extent of Förster resonance energy transfer (FRET) between two sites labeled with a FRET donor and acceptor. The relatively large Förster radii of traditional FRET methods (R0 > 20 Å) lead to a fairly low contribution of diffusion. We introduced short-distance FRET (sdFRET) where Dbo, an asparagine residue conjugated to 2,3-diazabicyclo[2.2.2]octane, acts as acceptor paired with donors, such as naphtylalanine (NAla), tryptophan, 5-l-fluorotryptophan, or tyrosine. The Förster radii are always close to 10 Å, which makes sdFRET highly sensitive to diffusional motion. We recently found indications that the FRET enhancement caused by diffusion depends symmetrically on the product of the radiative fluorescence lifetime of the donor and the diffusion coefficient. In this study, we varied this product by two orders of magnitude, using both donors of different lifetime, NAla and FTrp, as well as a varying viscogen concentration, to corroborate this statement. We demonstrate the consequences of this relationship in evaluating the impact of viscogenic coadditives on peptide dimensions.

scholarly journals Rapid in Vitro Assembly Dynamics and Subunit Turnover of FtsZ Demonstrated by Fluorescence Resonance Energy Transfer

2005 ◽  
Vol 280 (23) ◽  
pp. 22549-22554 ◽  
Author(s):  
Yaodong Chen ◽  
Harold P. Erickson
Keyword(s):  
Energy Transfer ◽  
Steady State ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Half Time ◽  
Gtp Hydrolysis ◽  
Donor And Acceptor ◽  
Fluorescence Resonance

We have developed an assay for the assembly of FtsZ based on fluorescence resonance energy transfer (FRET). We mutated an innocuous surface residue to cysteine and labeled separate pools with fluorescein (donor) and tetramethylrhodamine (acceptor). When the pools were mixed and GTP was added, assembly produced a FRET signal that was linearly proportional to FtsZ concentration from 0.7 μm (the critical concentration (Cc)) to 3 μm. At concentrations greater than 3 μm, an enhanced FRET signal was observed with both GTP and GDP, indicating additional assembly above this second Cc. This second Cc varied with Mg2+ concentration, whereas the 0.7 μmCc did not. We used the FRET assay to measure the kinetics of initial assembly by stopped flow. The data were fit by the simple kinetic model used previously: monomer activation, a weak dimer nucleus, and elongation, although with some differences in kinetic parameters from the L68W mutant. We then studied the rate of turnover at steady state by pre-assembling separate pools of donor and acceptor protofilaments. When the pools were mixed, a FRET signal developed with a half-time of 7 s, demonstrating a rapid and continuous disassembly and reassembly of protofilaments at steady state. This is comparable with the 9-s half-time for FtsZ turnover in vivo and the 8-s turnover time of GTP hydrolysis in vitro. Finally, we found that an excess of GDP caused disassembly of protofilaments with a half-time of 5 s. Our new data suggest that GDP does not exchange into intact protofilaments. Rather, our interpretation is that subunits are released following GTP hydrolysis, and then they exchange GDP for GTP and reassemble into new protofilaments, all on a time scale of 7 s. The mechanism may be related to the dynamic instability of microtubules.

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