Improved FRET-Based Trilateration Methods Applied to the Mapping of Calmodulin within the Ryanodine Receptor

Abstract

We have used improved trilateration methods, simulated annealing calculations, and time-resolved FRET measurements to map the location of calmodulin (CaM) within the ryanodine receptor (RyR) Ca2+ channel. Our previous published FRET trilateration method used distances from confocal microscopy measurements, where the distance uncertainties were calculated based on measurement precision (Svensson et al. 2014 Biophys J. 107, 2037-48). 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 new generic trilateration approach takes into account the Gaussian distance distribution and shows the FRET probability distributions as an MRC/CCP4-format density map, which 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. Five single-cysteine variants of the 12-kDa FK506-binding protein (FKBP) were labeled with FRET donor, Alexa Fluor 488, 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. Four single-cysteine variants of CaM, two N-lobe and two C-lobe sites, were labelled with a fluorescence acceptor, Alexa Fluor 568. FRET from the FKBP donors to the CaM acceptor was measured via FLT. FRET-based trilateration has allowed us to resolve both the location of the N- and C-lobes of CaM within the RyR cryo-EM map. A shift in the location of CaM dependent on resting or contracting [Ca2+] is observed. This work was supported by NIH grants R01 HL092097 (RLC), and R37 AG26160 (DDT) and by a Postdoctoral Fellowship from the American Heart Association (RTR).