Detecting stoichiometry of macromolecular complexes in live cells using FRET.

Manu Ben-Johny,David T Yue,Daniel N Yue

doi:10.1038/ncomms13709

Manu Ben-Johny, David T Yue + Show 1 more

Open Access

https://doi.org/10.1038/ncomms13709

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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.

Highlights

The stoichiometry of macromolecular interactions is fundamental to cellular signalling yet challenging to detect from living cells
We demonstrate the utility of this method by addressing the long-standing controversy of stoichiometry of CaM binding to voltage-gated ion channels
The process of Fluorescence resonance energy transfer (FRET) alters two key features of the total fluorescence emission spectrum of the bound donor-acceptor complex: (1) quenching of the fluorescence intensity of the donor and (2) increase in the fluorescence intensity of the acceptor[27,28] (Supplementary Fig. 1). These spectral changes imply that FRET efficiency can be determined using two distinct metrics[27]: (1) a donor-centric measure that reports the fractional reduction in the donor’s fluorescence intensity as a result of FRET29–32 and (2) an acceptor-centric measure that quantifies sensitized emission or the fractional enhancement in the acceptor’s fluorescence intensity due to FRET9,28,30,32–35

Summary

Results

If we consider FRET between CFP-tagged CaM and YFP-tagged full length myosin Va neck domain containing all six tandem IQ motifs (Fig. 3c,d), EA,max (0.255±0.005, mean±s.e.m.; n 1⁄4 26; Fig. 3d, left subpanel) is substantially larger than ED,max (0.043±0.007, mean±s.e.m.; n 1⁄4 44; Fig. 3d, right subpanel) These efficiencies yield a stoichiometry ratio, u 1⁄4 5.91±0.15 (mean±s.e.m.), a. These results demonstrate the strong correlation between the experimentally determined stoichiometry ratio (u) to the number of donor to acceptor molecules in the bound complex This outcome further corroborates the reliability and the flexibility of our FRET-based assay to determine the stoichiometry of binding interactions within live cells. Our findings illustrate the suitability and resolving power of our assay to discern dynamic changes in stoichiometry of signalling molecules within large macromolecular complexes such as ion channels

Discussion

CaM 1 CaM

Methods