NMR Studies of the Interaction of Calmodulin with IQ Motif Peptides
Calmodulin (CaM) is a ubiquitous EF-hand calcium sensor protein that transduces calcium signals in a wide range of signaling pathways. Structural analysis of complexes with peptides has provided valuable insights into the remarkable variety in the way in which CaM interacts with and activates its targets. Among these various targets, CaM has been shown to be an essential component of a calcium-sensing regulatory apparatus for a number of voltage-gated ion channels. NMR spectroscopy has proven to be a powerful tool for the structural characterization of CaM–peptide complexes, in particular for the study of IQ motifs, which bind CaM at the basal level of calcium in cells and thereby serve to localize CaM to its sites of action. We describe here methods for the robust expression and purification of CaM isotopically enriched for NMR analysis, as well as for the complex of CaM with a peptide derived from the IQ motif sequence of the human cardiac sodium channel NaV1.5. We also describe methods for NMR analysis of titrations of CaM with IQ motif peptides to determine the stoichiometry of the complex and to identify the residues at the binding interface.
Key wordsCalmodulin NaV1.5 IQ motif 15N-1H HSQC NMR spectroscopy Protein complex Co-expression Calcium binding protein Calcium signaling EF-hand
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