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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.

journal_name

Biophys J

journal_title

Biophysical journal

authors

Her C,McCaffrey JE,Thomas DD,Karim CB

doi

10.1016/j.bpj.2016.10.025

subject

Has Abstract

pub_date

2016-12-06 00:00:00

pages

2387-2394

issue

eissn

0006-3495

issn

1542-0086

pii

S0006-3495(16)30952-3

journal_volume

111

pub_type

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