Detecting stoichiometry of macromolecular complexes in live cells using FRET

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.

Quantifying the inhibitory effect of Bcl‐xl on the action of Mff using live‐cell fluorescence imaging

“…The stoichiometry ( v ) of a D–A complex is where E A,max (slope) was determined by linearly fitting the E D – R C plot, and E D,max (slope) was determined by linearly fitting the E A –1/ R C plot :…”

Backbone resonance assignments of complexes of apo human calmodulin bound to IQ motif peptides of voltage-dependent sodium channels NaV1.1, NaV1.4 and NaV1.7

“…CaM binds to a well-conserved IQ motif [IQxxx[R,K]Gxxx[R,K] found in the intracellular C-terminal domain of the α-subunit of all Na V isoforms (Mori et al 2000; Theoharis et al 2008; Sarhan et al 2012; Gabelli et al 2016; Hovey et al 2017; Kim et al 2004) and regulates the function of some isoforms in a calcium-dependent manner (Young and Caldwell 2005; Ben-Johny, Yue, and Yue 2016; Yan et al 2017; Pitt and Lee 2016). However, the mechanisms of calcium- and CaM-dependent regulation of channel function are complex and remain controversial.…”

Superior robustness of ExEm‐spFRET to IIem‐spFRET method in live‐cell FRET measurement

“…, ; Johny et al. , ). There are two key issues for quantitative FRET measurement especially in living cells: donor emission crosstalk (donor fluorescence is collected in acceptor detection channel) and acceptor excitation crosstalk (acceptor is excited directly under donor excitation) due to the spectral overlap of FPs.…”

“…Fluorescent proteins (FPs)-based fluorescence resonance energy transfer (FRET) has been widely used as a powerful technique to study protein-protein interaction (Chen et al, 2007;Dimura et al, 2016;Bunt & Wouters, 2017) and stoichiometry of macromolecular complexes in living cells (Johny et al, 2012;Kawano et al, 2013). Quantitative FRET measurement is essential for FRET two-hybrid assay (Butz et al, 2016;Johny et al, 2016). There are two key issues for quantitative FRET measurement especially in living cells: donor emission crosstalk (donor fluorescence is collected in acceptor detection channel) and acceptor excitation crosstalk (acceptor is excited Two-wavelengths excitation-based spectral linear unmixing of emission spectra (Em-unmixing) has been used for quantitative FRET measurement (spFRET) (Thaler et al, 2005;Wlodarczyk et al, 2008;Levy et al, 2011;Zhang et al, 2014;Zhang et al, 2016).…”