Bidirectional Fluorescence Resonance Energy Transfer (FRET) in Mutated and Chemically Modified Yellow Fluorescent Protein (YFP)

2011 ◽  
Vol 22 (2) ◽  
pp. 227-234 ◽  
Author(s):  
Bobin George Abraham ◽  
Nikolai V. Tkachenko ◽  
Ville Santala ◽  
Helge Lemmetyinen ◽  
Matti Karp
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Chemically Modified ◽  
Fluorescence Resonance

scholarly journals Ultra-Sensitive Hydrogen Peroxide Sensor Based on Peroxiredoxin and Fluorescence Resonance Energy Transfer

2020 ◽  
Vol 10 (10) ◽  
pp. 3508
Author(s):  
Haijun Yu ◽  
Haoxiang Li ◽  
Yao Zhou ◽  
Shengmin Zhou ◽  
Ping Wang
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Disulfide Bonds ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Mixed Disulfide ◽  
Fluorescence Resonance ◽  
Protein Sensor

In this paper, a fluorescence resonance energy transfer (FRET)-based sensor for ultra-sensitive detection of H2O2 was developed by utilizing the unique enzymatic properties of peroxiredoxin (Prx) to H2O2. Cyan and yellow fluorescent protein (CFP and YFP) were fused to Prx and mutant thioredoxin (mTrx), respectively. In the presence of H2O2, Prx was oxidized into covalent homodimer through disulfide bonds, which were further reduced by mTrx to form a stable mixed disulfide bond intermediate between CFP-Prx and mTrx-YFP, inducing FRET. A linear quantification range of 10–320 nM was obtained according to the applied protein concentrations and the detection limit (LOD) was determined to be as low as 4 nM. By the assistance of glucose oxidase to transform glucose into H2O2, the CFP-Prx/mTrx-YFP system (CPmTY) was further exploited for the detection of glucose in real sample with good performance, suggesting this CPmTY protein sensor is highly practical.

Abstract 16905: Fluorescence Resonance Energy Transfer Reveals that Cardiac Calcium ATPase Dimerizes and Forms a Complex with Phospholamban in a 2:1 Stoichiometry

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Daniel Blackwell ◽  
Seth L Robia
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Calcium Atpase ◽  
The Other ◽  
Fret Efficiency ◽  
Fluorescence Resonance

The sarco/endoplasmic reticulum calcium ATPase (SERCA) has been proposed to form functional dimers in vitro. In order to investigate whether SERCA forms homo-dimers in live cells, we fused canine SERCA2a to cerulean (Cer) or yellow fluorescent protein (YFP), and quantified SERCA-SERCA interactions by fluorescence resonance energy transfer (FRET). SERCA-SERCA FRET efficiency was dependent on the labeling position of the fluorescent protein tags, with the highest FRET efficiency achieved when the respective fluorescent proteins were fused to SERCA N-termini. FRET was reduced by competition with unlabeled SERCA, suggesting that the observed FRET was due to specific protein-protein interactions. Progressive photobleaching of YFP showed that Cer intensity increased linearly with decreasing YFP intensity, suggesting that the stoichiometry of the SERCA complex is a dimer. In contrast, a control experiment with phospholamban (PLB) oligomer showed a non-linear YFP/Cer relationship, consistent with its well-known pentameric stoichiometry. We also investigated whether SERCA dimers could interact with PLB, the regulatory binding partner of SERCA. Interestingly, while average maximal FRET was 28% between SERCA and PLB, fluorescence lifetime measurements revealed two different lifetimes, consistent with two different populations of FRET donors. One population showed very low FRET, while the other population exhibited high FRET- approximately double the measured average maximal FRET efficiency. The data are consistent with a single PLB bound to each SERCA homo-dimer; in this regulatory complex one SERCA protomer is in close proximity to PLB (50 Å), while the other is too far away to participate in FRET with PLB.

scholarly journals Mapping the Architecture of Secretin Receptors with Intramolecular Fluorescence Resonance Energy Transfer Using Acousto-Optic Tunable Filter-Based Spectral Imaging

2007 ◽  
Vol 21 (8) ◽  
pp. 1997-2008 ◽  
Author(s):  
Cayle S. Lisenbee ◽  
Kaleeckal G. Harikumar ◽  
Laurence J. Miller
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Tunable Filter ◽  
Intracellular Loop ◽  
Secretin Receptor ◽  
Fluorescence Resonance

Abstract The molecular structure and agonist-induced conformational changes of class II G protein-coupled receptors are poorly understood. In this work, we developed and characterized a series of dual cyan fluorescent protein (CFP)-tagged and yellow fluorescent protein (YFP)-tagged secretin receptor constructs for use in various functional and fluorescence analyses of receptor structural variants. CFP insertions within the first or second intracellular loop domains of this receptor were tolerated poorly or partially, respectively, in receptors tagged with a carboxyl-terminal yellow fluorescent protein that itself had no effect on secretin binding or cAMP production. A similar CFP insertion into the third intracellular loop resulted in a plasma membrane-localized receptor that bound secretin and signaled normally. This fully active third-loop variant exhibited a significant decrease in fluorescence resonance energy transfer signals that were recorded with an acousto-optic tunable filter microscope after exposure to secretin agonist but not to a receptor antagonist. These data demonstrate changes in the relative positions of intracellular structures that support a model for secretin receptor activation.

scholarly journals Use of Fluorescence Resonance Energy Transfer for Rapid Detection of Enteroviral Infection In Vivo

2006 ◽  
Vol 72 (5) ◽  
pp. 3710-3715 ◽  
Author(s):  
Yu-Chen Hwang ◽  
Wilfred Chen ◽  
Marylynn V. Yates
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Fluorescent Protein ◽  
Direct Method ◽  
Yellow Fluorescent Protein ◽  
Clinical Manifestations ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Genetically Engineered ◽  
Fluorescence Resonance

ABSTRACT Enteroviruses can be easily transmitted through the fecal-oral route and cause a diverse array of clinical manifestations. Recent outbreaks associated with enteroviral contamination in aquatic environments have called for the development of a more efficient and accurate virus monitoring system. To develop a simple, rapid, and direct method for identifying enteroviral infections, we generated a fluorescent reporter system in which genetically engineered cells express a hybrid fluorescent indicator composed of a linker peptide, which is exclusively cleaved by the 2A protease (2Apro), flanked with a cyan fluorescent protein (CFP) and a yellow fluorescent protein undergoing fluorescence resonance energy transfer. The covalent linkage between two fluorophores is disrupted due to 2Apro activity upon viral infection, which results in an increase in CFP intensity. This allows the rapid (within 7.5 h) detection of very low numbers (10 PFU or fewer) of infectious enteroviruses.

scholarly journals Cdc42, Rac1, and Rac2 Display Distinct Patterns of Activation during Phagocytosis

2004 ◽  
Vol 15 (8) ◽  
pp. 3509-3519 ◽  
Author(s):  
Adam D. Hoppe ◽  
Joel A. Swanson
Keyword(s):  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
G Proteins ◽  
Fluorescent Protein ◽  
Yellow Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Protein Activation ◽  
Poor Region ◽  
Fluorescence Resonance

The small G proteins Cdc42, Rac1, and Rac2 regulate the rearrangements of actin and membrane necessary for Fcγ receptor-mediated phagocytosis by macrophages. Activated, GTP-bound Cdc42, Rac1, and Rac2 bind to the p21-binding domain (PBD) of PAK1, and this interaction provided a basis for microscopic methods to localize activation of these G proteins inside cells. Fluorescence resonance energy transfer-based stoichiometry of fluorescent chimeras of actin, PBD, Cdc42, Rac1, and Rac2 was used to quantify G protein activation relative to actin movements during phagocytosis of IgG-opsonized erythrocytes. The activation dynamics of endogenous G proteins, localized using yellow fluorescent protein-labeled PBD, was restricted to phagocytic cups, with a prominent spike of activation over an actin-poor region at the base of the cup. Refinements of fluorescence resonance energy transfer stoichiometry allowed calculation of the fractions of activated GTPases in forming phagosomes. Cdc42 activation was restricted to the leading margin of the cell, whereas Rac1 was active throughout the phagocytic cup. During phagosome closure, activation of Rac1 and Rac2 increased uniformly and transiently in the actin-poor region of phagosomal membrane. These distinct roles for Cdc42, Rac1, and Rac2 in the component activities of phagocytosis indicate mechanisms by which their differential regulation coordinates rearrangements of actin and membranes.

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