CaM mutagenesis and purification

Tetra-Cys CaM was engineered containing two Cys in the N-lobe (T34CS38C) and C-lobe (R106CT110C) followed by cross linking with BSL to measure intra-molecular distances by DEER. Di-Cys CaM mutants (T34CS38C or R106CT110C) were also expressed and purified for experiments involving inter-protein distance measurement with TOAC labeled RyRp. In detail, recombinant human CaM was expressed in Escherichia coli using the pET-7 vector⁴⁷. Mutations targeting the Cys labeling sites were introduced using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA), and the mutation was verified by DNA sequencing. Following induction with isopropyl β-D-1-thiogalactopyraoside, di- and tetra-Cys-CaM were purified via phenyl-Sepharose chromatography⁴⁸. Protein concentrations were determined by the bicinchoninic acid procedure (Pierce, Rockford, IL) using bovine brain CaM as the standard⁴⁹.