title: 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.
authors.affiliation: Calcium Signals Laboratory, Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Ross Building, Room 713, 720 Rutland Avenue, Baltimore, Maryland 21205, USA.
authors.forename: Manu
authors.initials: M
authors.lastname: Ben-Johny
authors.affiliation: Calcium Signals Laboratory, Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Ross Building, Room 713, 720 Rutland Avenue, Baltimore, Maryland 21205, USA.
authors.forename: Daniel N
authors.initials: DN
authors.lastname: Yue
authors.affiliation: Calcium Signals Laboratory, Department of Biomedical Engineering, The Johns Hopkins University School of Medicine, Ross Building, Room 713, 720 Rutland Avenue, Baltimore, Maryland 21205, USA.
authors.forename: David T
authors.initials: DT
authors.lastname: Yue
mesh_terms: D000818:Animals; D015220:Calcium Channels / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002470:Cell Survival; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D046911:Macromolecular Substances / Q000378:metabolism*; D051379:Mice; D024701:Myosin Type V / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D000072417:Protein Domains; D015203:Reproducibility of Results; D015222:Sodium Channels / Q000378:metabolism
chemical_list: D015220:Calcium Channels; D002147:Calmodulin; D008164:Luminescent Proteins; D046911:Macromolecular Substances; D015222:Sodium Channels; D024701:Myosin Type V