Enrich sec 650 10 300 column

The stoichiometry of TevENO-Nb11 complex was determined by analytical SEC using an ENrich^TM SEC 650 10 × 300 Column (Bio-Rad), pre-equilibrated in buffer A. Samples of 500 μL containing 500 μg TevENO, 168.9 μg Nb11, or TevENO-Nb11 complex mixed at varying molar ratios (2:1, 2:2, 2:3, and 2:4), were injected onto the column and eluted at a flow rate of 1 mL min⁻¹. The TevENO-Nb11 complexes were mixed and incubated for 1 h prior to their application on the column. The column was calibrated with the BioRad molecular mass standard under the same conditions. The elution peaks of all chromatograms were analyzed by SDS-PAGE as described above.

Mitochondrial proteins (4 mg) were solubilized in 1.25 mL of 1X Native buffer (20 mM Tris, pH 7.0, 50 mM NaCl, 1 mM EDTA, 10% glycerol, 1 mM PMSF, and 1% digitonin). The solubilized proteins were loaded on a ENrich^TM SEC 650 10 × 300 column (Bio-Rad, Hercules, CA, USA) and run on an NGC system (BioRad). The proteins were eluted with the same buffer, except the digitonin concentration was reduced to 0.1%. After discarding the void volume (6.0 mL), 18 fractions (1.0 mL each) were collected. Proteins in each fraction were analyzed via SDS-PAGE and immunoblotting using antibodies for Myc, HA, TbTim17, VDAC, and mHsp70. Gel filtration molecular weight markers were run separately on the column under same conditions and were detected in the eluted fractions via SDS-PAGE and CB-staining (Supplementary Figure S1). The sizes of the small TbTims complexes and TbTim17 complexes were calculated from a standard curve, in which the log10 values of molecular sizes of marker proteins were plotted against the elution volumes.

Purified NhaA-GFP_8His fusion protein was diluted in buffer containing 150 mM NaCl, 20 mM Tris pH 7.5 and 0.03% (w/v) DDM to a final RFU of ~5000, which corresponds to concentration of 0.02 μg μL⁻¹. A 120 μL of the diluted sample was then aliquoted into duplicate 1.5 mL tubes containing increasing concentrations of cardiolipin 18:1 (Avanti, cat. no. 710335P), as well as into a control containing an equal volume of 1% (w/v) DDM instead of lipid. β-OG was then added into all the mixtures to a final concentration of 1% (w/v) and then mixed by pipetting. Samples were heated for 10 min at 47 °C and centrifuged at 18,000g using a Microfuge^® 18 Centrifuge at 4 °C. The GFP fluorescence of the supernatant was then measured using a plate reader as described in the previous section, the samples recovered, and then further injected onto a ENrich^TM SEC 650 10 × 300 Column (BIO-RAD) pre-equilibrated with 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.03% (w/v) DDM at a flow rate of 1 mL min⁻¹ at room temperature using an inline-detector Shimadzu HPLC system (Shimadzu Corporation). The CL dependent oligomerization concentrations were calculated with 19 different cardiolipin (18:1) concentrations that were fitted by nonlinear regression, and the values reported are the averaged mean ± s.e.m. of the fit from n = 3 independent titrations.