FRET-Based Trilateration of Probes Bound within Functional Ryanodine Receptors

摘要: To locate the biosensor peptide DPc10 bound to ryanodine receptor (RyR) Ca 2þ channels, we developed an approach that combines fluorescence resonance energy transfer (FRET), simulated-annealing, cryo-electron microscopy, and crystallographic data. is identical 2460-2495 segment within cardiac muscle RyR isoform (RyR2) central domain. binding RyR2 results in a pathologically elevated leak by destabilizing key interactions between N-terminal domains (unzipping). localize site RyR2, measured FRET five single-cysteine variants of FK506-binding protein (FKBP) labeled with donor probe, acceptor probe (A-DPc10). Effective positions were calculated from simulated-annealing constrained both cryo-EM map FKBP atomic structure docked RyR. A-DPc10 was permeabilized cardiomyo- cytes via confocal converted distances, used trilaterate locus Additional measurements donor-labeled calmodulin constrain trilaterations. Results domain 3, ~35 Au previously crystal structure. This multiscale may be useful mapping other sites mechanistic interest range FKBP.