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Type: Journal article
Title: Measurement of heterotrimeric G-protein and regulators of G-protein signaling interactions by time-resolved fluorescence resonance energy transfer
Author: Leifert, W.
Hill, K.
Cooper, T.
Aloia, A.
Glatz, R.
McMurchie, E.
Citation: Analytical Biochemistry, 2006; 355(2):201-212
Publisher: Academic Press Inc Elsevier Science
Issue Date: 2006
ISSN: 0003-2697
Statement of
Leifert, Wayne R.; Bailey, Kelly; Cooper, Tamara H.; Aloia, Amanda L.; Glatz, Richard V.; McMurchie, Edward J.
Abstract: G-protein-coupled receptors transduce their signals through G-protein subunits which in turn are subject to modulation by other intracellular proteins such as the regulators of G-protein signaling (RGS) proteins. We have developed a cell-free, homogeneous (mix and read format), time-resolved fluorescence resonance energy transfer (TR-FRET) assay to monitor heterotrimeric G-protein subunit interactions and the interaction of the G alpha subunit with RGS4. The assay uses a FRET pair consisting of a terbium cryptate chelate donor spectrally matched to an Alexa546 fluor acceptor, each of which is conjugated to separate protein binding partners, these being G alpha(i1):beta4gamma2 or G alpha(i1):RGS4. Under conditions favoring specific binding between labeled partners, high-affinity interactions were observed as a rapid increase (>fivefold) in the FRET signal. The specificity of these interactions was demonstrated using denaturing or competitive conditions which caused significant reductions in fluorescence (50-85%) indicating that labeled proteins were no longer in close proximity. We also report differential binding effects as a result of altered activation state of the G alpha(i1) protein. This assay confirms that interactions between G-protein subunits and RGS4 can be measured using TR-FRET in a cell- and receptor-free environment.
Keywords: Terbium
Organometallic Compounds
Quinolinium Compounds
Heterotrimeric GTP-Binding Proteins
RGS Proteins
Receptors, Cell Surface
Protein Subunits
Fluorescence Resonance Energy Transfer
Reproducibility of Results
Signal Transduction
Protein Binding
Time Factors
DOI: 10.1016/j.ab.2006.04.042
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