Affiliation:
1. Department of Physics, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin, United States
Abstract
Background:
The growing evidence that G protein-coupled receptors (GPCRs) not only form oligomers
but that the oligomers also may modulate the receptor function provides a promising avenue in the area of
drug design. Highly selective drugs targeting distinct oligomeric sub-states offer the potential to increase efficacy
while reducing side effects. In this regard, determining the various oligomeric configurations and geometric
sub-states of a membrane receptor is of utmost importance.
Methods:
In this report, we have reviewed two techniques that have proven to be valuable in monitoring the
quaternary structure of proteins in vivo: Fӧrster resonance energy transfer (FRET) spectrometry and fluorescence
intensity fluctuation (FIF) spectrometry. In FRET spectrometry, distributions of pixel-level FRET efficiency
are analyzed using theoretical models of various quaternary structures to determine the geometry and
stoichiometry of protein oligomers. In FIF spectrometry, spatial fluctuations of fluorescent molecule intensities
are analyzed to reveal quantitative information on the size and stability of protein oligomers.
Results:
We demonstrate the application of these techniques to a number of different fluorescence-based studies
of cells expressing fluorescently labeled membrane receptors, both in the presence and absence of various
ligands. The results show the effectiveness of using FRET spectrometry to determine detailed information regarding
the quaternary structure receptors form, as well as FIF and FRET for determining the relative abundance
of different-sized oligomers when an equilibrium forms between such structures.
Conclusion:
FRET and FIF spectrometry are valuable techniques for characterizing membrane receptor oligomers,
which are of great benefit to structure‐based drug design.
Funder
UWM Research Growth Initiative
National Science Foundation
Publisher
Bentham Science Publishers Ltd.
Subject
Drug Discovery,Pharmacology
Cited by
10 articles.
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