Punk instrument

Dynamic light scattering experiments were carried out using a pUNk instrument (Unchained Labs) according the manufacturer’s instructions. Purified protein (5 μl at 5 mg/ml) was loaded into a blade cell. Using an unbiased approach, the optimal traces from 10 runs were combined to calculate hydrodynamic diameters and polydispersity values.

The human cIAP2/XIAP-BIR1 domains were cloned in pET28b and expressed and purified, as already described [11 (link)]. Briefly, plasmids for cIAP2-BIR1 or XIAP-BIR1 expression were used to transform Escherichia coli strain BL21-CodonPlus (DE3)-RP competent cells (Agilent). After induction with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG, Sigma-Aldrich), bacteria were grown for 3 h at 37 °C. After cell lysis, the expressed proteins were purified using Ni-NTA affinity column (His-trap FFcrude, Cytiva) and size exclusion chromatography (Superdex 75, Cytiva). Proteins were concentrated with Amicon Ultra centrifugal filters (with 3 kDa cut-off) in buffer 50 mM Tris-HCl pH 8.0, 200 mM NaCl, 10 mM DTT. Dynamic Light Scattering assays were performed in pUNK instrument (Unchained Labs) at a concentration of 1 mg/mL and confirmed the high quality of the samples obtained, revealing a sample size polydispersity lower than 20% for all protein constructs and hydrodynamic radii comparable to the monomeric forms of the proteins.