In Vitro Fluorescence Resonance Energy Transfer-Based Assay Used to Determine the Rab27-Effector-Binding Affinity

2020 ◽  
Vol 18 (4) ◽  
pp. 180-194
Author(s):  
Raghdan Z. Al-Saad ◽  
Ian Kerr ◽  
Alistair N. Hume
Keyword(s):  
Energy Transfer ◽  
Binding Affinity ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Fluorescence Resonance ◽  
Effector Binding

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.

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.

A Light-Up Fluorescence Resonance Energy Transfer Magnetic Aptamer-Sensor for Ultra-Sensitive Lung Cancer Exosome Detection

2021 ◽  
Author(s):  
Nanhang Zhu ◽  
Guohao Li ◽  
Juan Zhou ◽  
Yujia Zhang ◽  
Ke Kang ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Lung Cancers ◽  
Reliable Detection ◽  
Fluorescence Resonance ◽  
Aptamer Sensor

In vitro liquid biopsy based on exosomes offers promising opportunities for fast and reliable detection of lung cancers. In this work, we present a fluorescence resonance energy transfer (FRET) magnetic...

scholarly journals Association of a Novel Preribosomal Complex in Trypanosoma brucei Determined by Fluorescence Resonance Energy Transfer

2012 ◽  
Vol 12 (2) ◽  
pp. 322-329 ◽  
Author(s):  
Lei Wang ◽  
Martin Ciganda ◽  
Noreen Williams
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Trypanosoma Brucei ◽  
Rna Binding ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
5S Rrna ◽  
Rna Recognition Motif ◽  
Fluorescence Resonance

ABSTRACT We have previously reported that the trypanosome-specific proteins P34 and P37 form a unique preribosomal complex with ribosomal protein L5 and 5S rRNA in the nucleoplasm. We hypothesize that this novel trimolecular complex is necessary for stabilizing 5S rRNA in Trypanosoma brucei and is essential for the survival of the parasite. In vitro quantitative analysis of the association between the proteins L5 and P34 is fundamental to our understanding of this novel complex and thus our ability to exploit its unique characteristics. Here we used in vitro fluorescence resonance energy transfer (FRET) to analyze the association between L5 and P34. First, we demonstrated that FRET can be used to confirm the association between L5 and P34. We then determined that the binding constant for L5 and P34 is 0.60 ± 0.03 μM, which is in the range of protein-protein binding constants for RNA binding proteins. In addition, we used FRET to identify the critical regions of L5 and P34 involved in the protein-protein association. We found that the N-terminal APK-rich domain and RNA recognition motif (RRM) of P34 and the L18 domain of L5 are important for the association of the two proteins with each other. These results provide us with the framework for the discovery of ways to disrupt this essential complex.

An upconversion nanoparticle-based fluorescence resonance energy transfer system for effectively sensing caspase-3 activity

The Analyst ◽  
2018 ◽  
Vol 143 (3) ◽  
pp. 761-767 ◽  
Author(s):  
Lin Liu ◽  
Hua Zhang ◽  
Daqian Song ◽  
Zhenxin Wang
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Caspase 3 ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Transfer System ◽  
Activity Detection ◽  
Sensing Platform ◽  
Fluorescence Resonance

An upconversion nanoparticle-based fluorescence resonance energy transfer sensing platform has been developed for the caspase-3 activity detection in vitro and in cells.

scholarly journals Fluorescence resonance energy transfer analysis of mitochondrial:lipid association in the porcine oocyte

Reproduction ◽  
2006 ◽  
Vol 132 (6) ◽  
pp. 829-837 ◽  
Author(s):  
R G Sturmey ◽  
P J O’Toole ◽  
H J Leese
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Oocyte Maturation ◽  
Lipid Droplets ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Metabolic Role ◽  
Molecular Scale ◽  
Fluorescence Resonance

The role of endogenous lipid in the provision of energy during in vitro maturation of immature porcine oocytes has been studied. Fluorescence resonance energy transfer (FRET) acceptor bleaching methods have been used to examine mitochondrial:lipid droplet co-localisation in live oocytes. FRET experiments demonstrate whether organelles are within the FRET-distance (i.e. 6–10 nm), thus showing true association on a molecular scale. Immature and in vitro-matured porcine oocytes were stained with Mitotracker Green (MTG; mitochondria) and Nile Red (NR; lipid droplets). The data indicated sufficient overlap between MTG emission and NR excitation to support a FRET reaction and that mitochondria and lipid droplets were sufficiently co-localised for a FRET reaction to occur. When NR-stained lipid droplets were specifically bleached, a significant increase in the MTG signal in stained mitochondria was observed (FRET efficiency, E=22.2 ± 3.18%). These results strongly suggest a metabolic role for lipid metabolism during oocyte maturation. This conclusion was reinforced by the use of inhibitors of fatty acid β-oxidation, methyl palmoxirate or mercaptoacetate, exposure to which during oocyte maturation led to developmental failure post-fertilisation. These data provide strong evidence that MTG and NR can act as a FRET pair and that in porcine oocytes, mitochondria and lipid droplets lie within 6–10 nm of each other, indicating association on a molecular scale. The findings also suggest that endogenous triglycerides play an important role in energy metabolism during porcine in vitro maturation.

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