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Using intrinsic tryptophan fluorescence to measure heterotrimeric G-protein activation

Robin E. Muller, David P. Siderovski, Adam J. Kimple University of North Carolina 07/2009

The fluorescent amino acid tryptophan changes its fluorescence intensity with conformational changes. The Gα subunit of G-protein coupled receptors (GPCR) undergoes such a drastic conformational change upon ligand binding. The responsible switch region of Gα contains a highly-conserved tryptophan residue that was used in this study to measure GPCR activation.

 

The response of increasing protein concentration upon activation was robustly reflected by the change in tryptophan fluorescence and reliably measured by a BMG LABTECH POLARstar® Omega. Using a protein concentration of 1 µM or higher the assay resulted in Z' values of >0.9.

 

The assay is a sensitive and high-quality means to measure G-protein activation without the use of radiolabeled nucleotides. Performing the assays with the POLARstar Omega 96-well plate reader with on-board injectors offers the advantage of automating the assays in triplicate on multiple Gα mutants or multiple modulators of spontaneous GDP release.

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