FRET-Based Trilateration of a Domain Peptide Bound within Functional Ryanodine Receptors in Cardiomyocytes

Abstract

We have used trilateration, the method of using distances to determine a location in space, simulated annealing calculations, and confocal FRET measurements in permeabilized rat myocytes to determine the topology of a modulatory interface within the ryanodine receptor (RyR) Ca2+ channel. A small peptide (∼4kDa, termed DPc10), corresponding to a RyR central stretch, hypothetically destabilizes a key interaction between RyR N-terminal and central domains to promote the open channel. We used DPc10 labeled with a small fluorophore, HiLyte Fluor 647, as FRET acceptor. Five single-cysteine variants of the 12 kDa FK506-binding protein (FKBP) were labeled with FRET donor, Alexa Fluor 488 C5 maleimide, and targeted to RyR. Effective average positions of the donors were calculated from simulated annealing, constrained by the RyR cryo-EM map and by the FKBP atomic structure. FRET from the FKBP donors to the DPc10 acceptor was measured via confocal microscopy, and the calculated distances were used to trilaterate the acceptor location within the RyR 3D map. The trilateration method uses the distances determined by FRET from each donor's calculated average position, where the ranges of the distances were based on measurement precision for each donor labeling site. The DPc10-bound acceptor locus is found at a region in space matching four distance ranges from FRET, and was further constrained by FRET between donor-labeled calmodulin and F-DPc10. This locus is near the RyR N-terminal domain structure docked into the cryo-EM reconstructed map of RyR. Computational resources were provided by the Minnesota Supercomputing Institute. This work was supported by NIH grants R01HL092097 (to D.M.B. and R.L.C.), and R01GM27906 (to D.D.T.).