Ben-Johny, M. et al. Nat. Commun. 7, 13709 (2016).

Determining protein stoichiometry in complexes is a long-standing challenge in biochemistry, and numerous in vitro approaches are useful for making such measurements. However, measuring complex stoichiometry in living cells represents a much more challenging problem. Ben-Johny et al. address this challenge using a FRET-based approach that exploits the fact that donor- and acceptor-centric metrics of FRET efficiency are differentially affected by the ratio of donor to acceptor molecules in a complex. Comparing the two FRET efficiencies allows the relative stoichiometry of two proteins to be determined. The researchers validated the method on concatemers of fluorescent proteins with known composition and used the method to study a long-standing question regarding calmodulin binding to ion channels under high and low Ca2+ levels.