Chemical cross-linking of proteins

Purified ChlH was desalted into activation buffer (100 mM MES, 0.5 M NaCl, pH 6) using a Zeba Spin column according to the manufacturer's instructions. 1 mg ml⁻¹ of ChlH in 100 µl activation buffer was incubated with 2 mM 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC, Thermo Fisher) and 5 mM N-hydroxysuccinimide (NHS, Sigma–Aldrich) at room temperature for 15 min and then buffer exchanged into PBS. 1 mg ml⁻¹ activated ChlH was mixed with 1 mg ml⁻¹ ChlD and incubated at 34°C for 2 h prior to flash-freezing samples.

Cross-linked samples were analysed by SDS–PAGE using NuPAGE 3–8% Tris–acetate gels in denaturing buffer (Tris–glycine-SDS), and cross-linked protein detected by immunoblotting for Synechocystis ChlD and ChlH, using the methods reported above.