Structural Dynamics of Calmodulin using Bifunctional Spin Labels and Double Electron-Electron Resonance

Abstract

We have used site-directed spin labeling and double electron-electron resonance spectroscopy (DEER) to probe the structural dynamics of calmodulin in the context of regulating the skeletal muscle Ca release channel, the ryanodine receptor (RyR1). Conventional spin labels that attach using a single flexible di-sulfide linkage allow local spin label motion that limits the resolution and accuracy of distance or orientation measurements. To avoid this problem, we use a bifunctional spin label (BSL) that provides stereospecific attachment at Cys sites four residues apart on an α helix. A mutant calmodulin construct containing bifunctional labeling sites on helices in the N-lobe (T34CS38C) and in the C-lobe (R106CT110C) was expressed and labeled with BSL. DEER was used to measure the spin-spin distance distribution in the presence and absence of Ca. Our results show that in the absence of Ca, CaM is predominantly in one structure with a center distance of 4.22 nm, which is in in good agreement with the predicted structure based on the published NMR solution structure (4.30 nm). In the presence of Ca, CaM takes on two structures with center distances at 2.73 (34%) and 4.11 nm (66%). Neither of these distances agrees with that predicted by the x-ray crystal structure of Ca-CaM (5.6 nm). This discrepancy may be due to artifacts from the crystalline environment in x-ray, whereas our DEER measurement was made in frozen solution. In conclusion, these results indicate the potential of BSL for accurate distance measurement and suggest new structures of Ca-bound CaM in solution. Ongoing studies are expanding these experiments to CaM bound to a synthetic peptide that is part of the CaM-binding domain on RyR1. This work was supported by NIH grants to DDT (R37 AG26160, T32 AR007612).