FRET-based Trilateration Resolves Distinct Structural States and Transitions of Calmodulin Bound to Ryanodine Receptors

We used fluorescence lifetime FRET (FLT-FRET) and trilateration to map the location of calmodulin (CaM) bound to skeletal and cardiac ryanodine receptor (RyR) Ca2+ channels at resting vs. peak contracting [Ca2+] (nM vs. μM). Eight single-Cys variants of the 12.6-kDa FK506-binding protein (FKBP) were labeled with FRET donor (AlexaFluor 488), and targeted to RyR1 and RyR2. Four single-Cys variants of WT-CaM or the Ca2+-insensitive CaM1234, two N-lobe and two C-lobe sites, were labelled with acceptor (AlexaFluor 568).