Detecting stoichiometry of macromolecular complexes in live cells using FRET

Manu Ben-Johny (), Daniel N. Yue and David T. Yue
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Manu Ben-Johny: Calcium Signals Laboratory, The Johns Hopkins University School of Medicine
Daniel N. Yue: Calcium Signals Laboratory, The Johns Hopkins University School of Medicine
David T. Yue: Calcium Signals Laboratory, The Johns Hopkins University School of Medicine

Nature Communications, 2016, vol. 7, issue 1, 1-10

Abstract: Abstract The stoichiometry of macromolecular interactions is fundamental to cellular signalling yet challenging to detect from living cells. Fluorescence resonance energy transfer (FRET) is a powerful phenomenon for characterizing close-range interactions whereby a donor fluorophore transfers energy to a closely juxtaposed acceptor. Recognizing that FRET measured from the acceptor’s perspective reports a related but distinct quantity versus the donor, we utilize the ratiometric comparison of the two to obtain the stoichiometry of a complex. Applying this principle to the long-standing controversy of calmodulin binding to ion channels, we find a surprising Ca2+-induced switch in calmodulin stoichiometry with Ca2+ channels—one calmodulin binds at basal cytosolic Ca2+ levels while two calmodulins interact following Ca2+ elevation. This feature is curiously absent for the related Na channels, also potently regulated by calmodulin. Overall, our assay adds to a burgeoning toolkit to pursue quantitative biochemistry of dynamic signalling complexes in living cells.

Date: 2016
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DOI: 10.1038/ncomms13709

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