Resonance energy transfer as a direct monitor of GTP-binding protein-effector interactions: activated .alpha.-transducin binding to the cGMP phosphodiesterase in the bovine phototransduction cascade

Biochemistry ◽  
1991 ◽  
Vol 30 (29) ◽  
pp. 7112-7118 ◽  
Author(s):  
Jon W. Erickson ◽  
Richard A. Cerione
Keyword(s):  
Energy Transfer ◽  
Binding Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Gtp Binding Protein ◽  
Cgmp Phosphodiesterase ◽  
Gtp Binding ◽  
Phototransduction Cascade

Homogeneous GTP Binding Assay Employing QRET Technology

2010 ◽  
Vol 15 (3) ◽  
pp. 261-267 ◽  
Author(s):  
Anita Rozwandowicz-Jansen ◽  
Jonne Laurila ◽  
Eija Martikkala ◽  
Heini Frang ◽  
Ilkka Hemmilä ◽  
...  
Keyword(s):  
Energy Transfer ◽  
G Protein ◽  
Binding Assay ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Fluorescent Detection ◽  
Time Resolved ◽  
Gtp Binding ◽  
Gtpγs Binding

Functional cell signaling assays have become important tools for measuring ligand-induced receptor activation in cell-based biomolecular screening. Guanosine-5′-triphosphate (GTP) is a generic signaling marker responsible for the first intracellular signaling event of the G-protein-coupled receptors (GPCRs). [35S]GTPγS binding assay is the classical well-established method for measuring agonist-induced G-protein activation requiring a separation of free and bound fractions prior to measurement. Here a novel, separation-free, time-resolved fluorescence GTP binding assay has been developed based on a non–fluorescence resonance energy transfer (FRET) single-label approach and quenching of a nonbound europium-labeled, nonhydrolyzable GTP analog (Eu-GTP). The quenching resonance energy transfer (QRET) method relies on the use of Eu-GTP, providing a time-resolved fluorescent detection as an alternative to the radiolabel [35S]GTPγS assay. Upon activation of recombinant human α2A-adrenoceptors (α2A-AR) expressed in Chinese hamster ovary cells, guanosine-5′-diphosphate is released from the α-subunit of Gi-proteins, enabling the subsequent binding of Eu-GTP. Activation of α2A-AR with 5 different α2-AR agonists was measured quantitatively using the developed QRET GTP assay and compared to [35S]GTPγS and heterogeneous Eu-GTP filtration assays. Equal potencies and efficacy rank orders were observed in all 3 assays but with a lower signal-to-background ratio and increased assay variation in the QRET assay compared to the Eu-GTP filtration and the nonhomogeneous [35S]GTPγS binding assays.

scholarly journals Design and Application of Highly Responsive Fluorescence Resonance Energy Transfer Biosensors for Detection of Sugar in Living Saccharomyces cerevisiae Cells

2007 ◽  
Vol 73 (22) ◽  
pp. 7408-7414 ◽  
Author(s):  
Jae-Seok Ha ◽  
Jae Jun Song ◽  
Young-Mi Lee ◽  
Su-Jin Kim ◽  
Jung-Hoon Sohn ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Energy Transfer ◽  
Fluorescence Resonance Energy Transfer ◽  
Signal Intensity ◽  
Binding Proteins ◽  
Dynamic Range ◽  
Binding Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy

ABSTRACT A protein sensor with a highly responsive fluorescence resonance energy transfer (FRET) signal for sensing sugars in living Saccharomyces cerevisiae cells was developed by combinatorial engineering of the domain linker and the binding protein moiety. Although FRET sensors based on microbial binding proteins have previously been created for visualizing various sugars in vivo, such sensors are limited due to a weak signal intensity and a narrow dynamic range. In the present study, the length and composition of the linker moiety of a FRET-based sensor consisting of CFP-linker1-maltose-binding protein-linker2-YFP were redesigned, which resulted in a 10-fold-higher signal intensity. Molecular modeling of the composite linker moieties, including the connecting peptide and terminal regions of the flanking proteins, suggested that an ordered helical structure was preferable for tighter coupling of the conformational change of the binding proteins to the FRET response. When the binding site residue Trp62 of the maltose-binding protein was diversified by saturation mutagenesis, the Leu mutant exhibited an increased binding constant (82 μM) accompanied by further improvement in the signal intensity. Finally, the maltose sensor with optimized linkers was redesigned to create a sugar sensor with a new specificity and a wide dynamic range. When the optimized maltose sensors were employed as in vivo sensors, highly responsive FRET images were generated from real-time analysis of maltose uptake of Saccharomyces cerevisiae (baker's yeast).

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