2.1. Preparation of untagged cTnC–cTnI chimera: cTnC(1–90)-C35S-C84S–cTnI(136–163)

A clone was prepared that included the N-terminal domain of human cardiac troponin C (residues 1–90 with C35S and C84S mutations) linked to the C-terminal region of troponin I (residues 136–163), based on the NMR chimera construct (Cai et al., 2016 ▸), in pET-28 vector (Table 1 ▸). The clone was expressed in Escherichia coli BL21 (DE3) cells (Novagen) at 37°C in LB medium to an OD₆₀₀ of 1.0 and was then induced using 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) at 37°C for 3 h.

All steps were performed at 4°C using an ÄKTA pure M1. Frozen cells harvested from 2 l of culture were suspended in 50 ml lysis buffer [20 mM Tris pH 7.5, 300 mM NaCl, 10 mM MgSO₄, 1 mM CaCl₂, 1 mM DTT, one tablet of cOmplete EDTA-free protease-inhibitor cocktail (Roche Applied Science) and 20 units per millilitre of benzonase nuclease (EMD Millipore)]. The cells were lysed by sonication. The lysate was clarified by sedimentation at 30 000g for 30 min (Thermo F21-8x50y rotor) and the supernatant was diluted sixfold in buffer A (20 mM Tris pH 7.5, 1 mM DTT, 1 mM CaCl₂) and loaded onto a 5 ml HiTrap Q FF (GE Healthcare) column equilibrated with buffer A. The column was washed with ten volumes of buffer A and the protein was eluted with a linear gradient of 0–700 mM NaCl in buffer A. Fractions were pooled and loaded onto a HiLoad 26/60 Superdex 200 column (GE Healthcare) equilibrated with buffer B (20 mM Tris pH 7.5, 300 mM NaCl, 0.1 mM CaCl₂, 1 mM DTT). The protein eluted as a monomer. The final yield of purified cTnC(1–90)-C35S-C84S–cTnI(136–163) protein was 80 mg per litre of culture. The protein purity was ≥98% as determined by SDS–PAGE analysis and Coomassie Brilliant Blue staining (data not shown). Electrospray ionization mass spectrometry confirmed the identity of the protein. Purified cTnC(1–90)-C35S-C84S–cTnI(136–163) was spiked with 1 M CaCl₂ to give a final concentration of 10 mM CaCl₂ and concentrated to 41.6 mg ml⁻¹ in 20 mM Tris pH 7.5, 300 mM NaCl, 10 mM CaCl₂, 1 mM DTT. Crystals were grown at 20°C in sitting drops from 1 µl protein solution mixed with 1 µl well solution consisting of 0.1 M sodium acetate pH 4.5, 0.2 M ammonium acetate, 16–20% PEG 4K. The crystals appeared spontaneously within 1–2 weeks. Seeding with previously obtained crystals improved the reproducibility and the speed of crystal formation. The crystals were flash-cooled in liquid nitrogen in cryosolution consisting of 75% well solution and 25% ethylene glycol.