Calcium-Dependent Structural Dynamics of a Spin-Labeled RyR Peptide Bound to Calmodulin

Abstract

We have used chemical synthesis, electron paramagnetic resonance (EPR), and circular dichroism to detect and analyze the structural dynamics of a ryanodine receptor (RyR) peptide bound to calmodulin (CaM). The skeletal muscle calcium release channel RyR1 is activated by Ca²⁺-free CaM and inhibited by Ca²⁺-bound CaM. To probe the structural mechanism for this regulation, wild-type RyRp and four spin-labeled derivatives were synthesized, each containing the nitroxide probe 2,2,6,6-tetramethyl-piperidine-1-oxyl-4-amino-4-carboxylic acid substituted for a single amino acid. In 2,2,6,6-tetramethyl-piperidine-1-oxyl-4-amino-4-carboxylic acid, the probe is rigidly and stereospecifically coupled to the α-carbon, enabling direct detection by EPR of peptide backbone structural dynamics. In the absence of CaM, circular dichroism indicates a complete lack of secondary structure, while 40% trifluoroethanol (TFE) induces >90% helicity and is unperturbed by the spin label. The EPR spectrum of each spin-labeled peptide indicates nanosecond dynamic disorder that is substantially reduced by TFE, but a significant gradient in dynamics is observed, decreasing from N- to C-terminus, both in the presence and absence of TFE. When bound to CaM, the probe nearest RyRp's N-terminus shows rapid rotational motion consistent with peptide backbone dynamics of a locally unfolded peptide, while the other three sites show substantial restriction of dynamics, consistent with helical folding. The two N-terminal sites, which bind to the C-lobe of CaM, do not show a significant Ca²⁺-dependence in mobility, while both C-terminal sites, which bind to the N-lobe of CaM, are significantly less mobile in the presence of bound Ca²⁺. These results support a model in which the interaction of RyR with CaM is nonuniform along the peptide, and the primary effect of Ca²⁺ is to increase the interaction of the C-terminal portion of the peptide with the N-terminal lobe of CaM. These results provide, to our knowledge, new insight into the Ca²⁺-dependent regulation of RyR by CaM.