2.5. Surface plasmon resonance (SPR) binding studies  

Recombinant DNA for full-length ClpC1 (ClpC1-FL) and ClpC1-NTD was codon-optimized, synthesized and cloned into pET-15b vector with an N-terminal His₆-SUMO tag (GenScript, Piscataway, New Jersey, USA). ClpC1-FL mutants V14A, Q17A, K85A and L92S/L96P and the N-terminal mutants FER (residues 2–145), AAFER, MAFER, MVFER and MVAFER also had His₆-SUMO N-terminal tags. All proteins were prepared using Ni–NTA mini spin columns (Qiagen, Germantown, Maryland, USA). The His₆-SUMO tags were cleaved with SUMO protease prepared in-house and the cleaved proteins were separated from their tags and the SUMO protease using Ni–NTA columns. The purified proteins were then buffer-exchanged into phosphate-buffered saline with 15% glycerol using a desalting column before storage at −80°C.

SPR studies were carried out using either a Biacore T200 or a Biacore 8K as reported previously (Wolf et al., 2019 ▸). In brief, ClpC1 proteins were immobilized on a CM5 sensor chip using standard amine coupling. RUF-I, ECU, OMS-A and OMS-B were isolated as described previously (Gao et al., 2014 ▸; Choules et al., 2019 ▸; Hur et al., 2018 ▸). ECU, RUF-I and OMS-A solutions were prepared at a series of increasing concentrations and were injected onto both blank surfaces and ClpC1 protein immobilized surfaces at a flow rate of 30 µl min⁻¹ at 25°C. All sensorgrams were double-referenced with blank channels and zero concentration, followed by fitting the data with two kinetic models (1:1 Langmuir and heterogeneous ligand models) using either Biacore T200 Evaluation Software version 3 or Biacore 8K Insight Evaluation Software. All experiments, excluding the N-terminal mutants, were carried out with both ClpC1-NTD and ClpC1-FL and similar results were obtained; therefore, just one set of data is provided for simplicity.