Detecting stoichiometry of macromolecular complexes in live cells using FRET

• Published in: Nature communications Vol. 7; no. 1; p. 13709
• Main Authors: Ben-Johny, Manu, Yue, Daniel N., Yue, David T.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 06.12.2016; Nature Publishing Group; Nature Portfolio
• Subjects: 14/33; 14/35; 631/1647/527/2047; 631/45/269/1146; 631/57/2267; 631/57/2270/1140; Animals; Calcium Channels - metabolism; Calmodulin - metabolism; Cell Survival; Energy; Fluorescence Resonance Energy Transfer; HEK293 Cells; Humanities and Social Sciences; Humans; Luminescent Proteins - metabolism; Macromolecular Substances - metabolism; Mice; multidisciplinary; Myosin Type V - chemistry; Myosin Type V - metabolism; Protein Binding; Protein Domains; Proteins; Reproducibility of Results; Science; Science (multidisciplinary); Sodium Channels - metabolism
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/ncomms13709

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 Ca 2+ -induced switch in calmodulin stoichiometry with Ca 2+ channels—one calmodulin binds at basal cytosolic Ca 2+ levels while two calmodulins interact following Ca 2+ 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. Measuring the in vivo stoichiometry of protein-protein interactions is challenging. Here the authors take a FRET-based approach, quantifying stoichiometry based on ratiometric comparison of donor and acceptor fluorescence, and apply their method to report on a Ca2 + -induced switch in calmodulin binding to Ca 2+ ion channels.