Synthesis of a new pair of fluorescence resonance energy transfer donor and acceptor dyes and its use in a protease assay

2005 ◽  
pp. 5459 ◽  
Author(s):  
Eva Katharina Kainmüller ◽  
Eulàlia Pinyol Ollé ◽  
Willi Bannwarth
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Donor And Acceptor ◽  
Fluorescence Resonance ◽  
Protease Assay

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.

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.

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 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.

scholarly journals Conformational Transitions of Polymer Chains in Solutions Characterized by Fluorescence Resonance Energy Transfer

Polymers ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 1007 ◽  
Author(s):  
Linlin Qin ◽  
Linling Li ◽  
Ye Sha ◽  
Ziyu Wang ◽  
Dongshan Zhou ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Conformational Changes ◽  
Conformational Transition ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Polymer Chains ◽  
Donor And Acceptor ◽  
Transition Concentration ◽  
Fluorescence Resonance

The critical overlap concentration C* is an important concept in polymer solutions and is defined as the boundary between dilute and semidilute regimes. In this study, the chain conformational changes of polystyrene (PS) with both high (Mn = 200,000 Da) and low (Mn = 13,000 Da) molecular weights in cis-decalin were compared by intrachain fluorescence resonance energy transfer (FRET). The random labeling of donor and acceptor chromophores strategy was employed for long PS chains, whereas chain-end labeling was used for short PS chains. By monitoring the spectroscopic intensity ratio between acceptor and donor, the concentration dependence on chain conformation from dilute to semidilute solutions was determined. Both long and short chains exhibit a conformational transition concentration, above which the polymer chains begin to collapse with concentration significantly. Interestingly, for randomly labeled polymer long chains, such concentration is consistent with C* determined from the viscosity result, below which only slight conformational change of polymer chain takes place. However, for the chain-end labeled short chain, the conformational transition concentration takes place earlier than C*, below which no significant polymer conformation change is observed.

Highly Adaptable and Sensitive Protease Assay Based on Fluorescence Resonance Energy Transfer

2011 ◽  
Vol 83 (19) ◽  
pp. 7356-7363 ◽  
Author(s):  
Thomas Zauner ◽  
Renate Berger-Hoffmann ◽  
Katrin Müller ◽  
Ralf Hoffmann ◽  
Thole Zuchner
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluorescence Resonance ◽  
Protease Assay

scholarly journals Spatial Distribution Analysis of AT- and GC-rich Regions in Nuclei Using Corrected Fluorescence Resonance Energy Transfer

2003 ◽  
Vol 51 (7) ◽  
pp. 951-958 ◽  
Author(s):  
Shin-ichi Murata ◽  
Petr Herman ◽  
Kunio Mochizuki ◽  
Tadao Nakazawa ◽  
Tetsuo Kondo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Spatial Distribution ◽  
Energy Transfer ◽  
Texture Analysis ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Distribution Analysis ◽  
Donor And Acceptor ◽  
Fluorescence Resonance

We employed microscopic intensity-based fluorescence resonance energy transfer (FRET) images with correction by donor and acceptor concentrations to obtain unbiased maps of spatial distribution of the AT- and GC-rich DNA regions in nuclei. FRET images of 137 bovine aortic endothelial cells stained by the AT-specific donor Hoechst 33258 and the GC-specific acceptor 7-aminoactinomycin D were acquired and corrected for the donor and acceptor concentrations by the Gordon's method based on the three fluorescence filter sets. The corrected FRET images were quantitatively analyzed by texture analysis to correlate the spatial distribution of the AT- and GC-rich DNA regions with different phases of the cell cycle. Both visual observation and quantitative texture analysis revealed an increased number and size of the low FRET efficiency centers for cells in the G2/M-phases, compared to the G1-phase cells. We have detected cell cycle-dependent changes of the spatial organization and separation of the AT- and GC-rich DNA regions. Using the corrected FRET (cFRET) technique, we were able to detect early DNA separation stages in late interphase nuclei.

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