FRET-Based Mapping of Calmodulin within the Ryanodine Receptor

Abstract

Using time-resolved fluorescence resonance energy transfer (TR-FRET) and multi-site-directed labeling of calmodulin (CaM) and FKBP12.6, we further resolve the structural basis underlying CaM regulation of skeletal and cardiac ryanodine receptor isoforms (RyR1 and RyR2) at relaxed and contracting [Ca2+]. Although cryo-EM and FRET-based mapping place Ca2+-bound CaM in a similar location on RyR1 and RyR2, the position of Ca2+-free (apo) CaM remains controversial - cryo-EM suggests a large shift in the centers of mass between apo- and Ca-CaM states, but this was not observed in previous FRET studies. Using TR-FRET, we resolved structural states of CaM bound to RyR1 and RyR2 in isolated sarcoplasmic reticulum membranes at 0.03 and 30 micromolar Ca2+. Donor fluorophores were targeted to the cytoplasmic assembly of RyR1 or RyR2 by pre-incubating sarcoplasmic reticulum vesicles with fluorescent FKBP12.6 labeled at one of five well dispersed sites (D-FKBP). FRET was measured in the presence of CaM labeled with an acceptor fluorophore attached to one of four sites (A-CaM) for each distance measurement. The donor-acceptor distances, calculated from fluorescence decay waveforms, were used to trilaterate the acceptor probe loci within RyR. On both RyR1 and RyR2, we detect a Ca2+-driven decrease in separation between CaM lobes, suggesting a more compact Ca2+-bound CaM, relative to apo-CaM, structure. However, these Ca2+-driven shifts in fluorophore positions are not as dramatic as the large shift previously proposed. We also observe isoform specific differences, as CaM's binding location appears to be rotated down and slightly inward on RyR2 relative to RyR1. This work was supported by NIH grants R01HL092097 (to RLC/DMB) and R37AG26160 (to DDT), and by a Postdoctoral Fellowship from the American Heart Association 16POST31010019 (to RTR).