Poly(A)-targeting molecular beacons: Fluorescence resonance energy transfer-based in vitro quantitation and time-dependent imaging in live cells

2012 ◽  
Vol 429 (2) ◽  
pp. 92-98 ◽  
Author(s):  
Min Young Kim ◽  
Jisu Kim ◽  
Sang Soo Hah
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Molecular Beacons ◽  
Time Dependent ◽  
Live Cells ◽  
Fluorescence Resonance

Analisis of A-Kinase Anchoring Protein Interactions With PKA Using Fluorescence Resonance Energy Transfer

2000 ◽  
Vol 6 (S2) ◽  
pp. 828-829
Author(s):  
M. L. Ruehr ◽  
D. S. Damron ◽  
M. Bond
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Regulatory Subunit ◽  
Live Cells ◽  
Human Thyroid ◽  
Local Concentration ◽  
Fluorescence Resonance

The clustering of components of a signaling pathway at a specific subcellular location raises the local concentration of the appropriate messengers and serves to amplify the signal. The cAMP dependent-protein kinase (PKA) pathway is regulated by compartmentalization of its components. A-kinase anchoring proteins (AKAPs) tether PKA to specific subcellular sites, thus presumably increasing substrate specificity. Phosphorylation of the type II regulatory subunit of PKA (RII) increases its affinity for AKAPs in vitro (1). The purpose of this study was to investigate whether altering the phosphorylation state of RII in live cells changes its affinity for an AKAP. Specifically, we investigated the binding kinetics between Ht31, a peptide containing the PKA binding portion of an AKAP from human thyroid (2), and RII, in response to PKA activators or inhibitors.Fluorescence resonance energy transfer (FRET) was used to monitor binding events between RII and the catalytic subunit (C) of PKA, Ht31, or Ht31P, a mutated form of Ht31 which does not bind RII.

scholarly journals Spectral wide-field microscopic fluorescence resonance energy transfer imaging in live cells

2015 ◽  
Vol 20 (8) ◽  
pp. 086011 ◽  
Author(s):  
Lili Zhang ◽  
Guiqi Qin ◽  
Liuying Chai ◽  
Jiang Zhang ◽  
Fangfang Yang ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Live Cells ◽  
Wide Field ◽  
Fluorescence Resonance

Single-molecule fluorescence resonance energy transfer techniques on rotary ATP synthases

2011 ◽  
Vol 392 (1-2) ◽  
Author(s):  
Michael Börsch
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Single Molecule ◽  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Single Molecule Fret ◽  
Intensity Changes ◽  
Fluorescence Resonance

Abstract Conformational changes of proteins can be monitored in real time by fluorescence resonance energy transfer (FRET). Two different fluorophores have to be attached to those protein domains which move during function. Distance fluctuations between the fluorophores are measured by relative fluorescence intensity changes or fluorescence lifetime changes. The rotary mechanics of the two motors of FoF1-ATP synthase have been studied in vitro by single-molecule FRET. The results are summarized and perspectives for other transport ATPases are discussed.

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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