FRET-Based Trilateration Applied to the Mapping of CaM within the Ryanodine Receptor

Abstract

We have carried out trilaterations, based on simulated annealing calculations and time-resolved FRET measurements, to map the location of calmodulin (CaM) within the ryanodine receptor (RyR) Ca2+ channel. Fluorescence lifetime (FLT) detection of FRET allows resolving multiple distances for each FRET pair, which are represented by Gaussian distance distributions fitted to the FRET data. Our improved general trilateration approach takes into account the distance distributions from multiple FRET pairs, and shows the location of the acceptor probe as a probability distribution in the format of an MRC/CCP4-format density map. This map can be displayed using common molecular graphics software. A more specialized approach takes into account the four-fold symmetry of the RyR channels in calculating the predicted FRET from a donor with known location to symmetrically located FRET acceptors. Several single-cysteine variants of the 12-kDa FK506-binding protein (FKBP) were labeled with FRET donor (Alexa Fluor 488), and targeted to RyR1 and RyR2. Effective average positions of the donors were calculated from simulated annealing, constrained by the FKBP atomic structure and by the RyR1 and RyR2 cryo-EM maps. Four single-cysteine variants of CaM, two N-lobe and two C-lobe sites, were labelled with fluorescence acceptor (Alexa Fluor 568). FRET from the FKBP donors to the CaM acceptor was measured via FLT detection. The FRET-based trilateration has allowed us to resolve the location of the N- and C-lobes of CaM within the RyR1 and RyR2 cryo-EM maps. A shift in the location of CaM dependent on resting or contracting [Ca2+] is observed. The upgraded methodology is setup for determining the structure-function relationship of other RyR modulators. This work was supported by NIH grants R01 HL92097 (RLC) and R37 AG26160 (DDT), and by American Heart Association Postdoctoral Fellowship 16POST31010019 (RTR).