Dynamics v7.8.1.3 software

A Dynapro Nanostar (Wyatt) equilibrated to 20°C was used for all DLS experiments. Protein samples at ~0.5 mg/mL in 20 mM Tris, 150 mM NaCl, 1 mM DTT, 10 mM CHAPS, pH 8.0 without or with 5 mM CaCl₂, 5 mM MgCl₂, 5 mM CoCl₂ or 0.5 mM CPACC were centrifuged at 12,000 ×g for 5 min before a 5 μL aliquot of the supernatant was removed and loaded into a JC501 cuvette (Wyatt). The sample was equilibrated for 5 min at 20°C before 10 autocorrelation function acquisitions of 5s each were recorded and averaged. Two aliquots (technical replicates) from each sample were averaged, and each experimental condition was performed on three independent/individual protein expression preparations (biological replicates). Autocorrelation functions were deconvoluted using the regularization algorithm to extract polydisperse distributions of hydrodynamic radii and cumulants algorithm to extract weight-averaged monodisperse hydrodynamic radii, using the accompanying Dynamics (v7.8.1.3) software (Wyatt). The dependence of hydrodynamic size on CPACC concentration was determined using ~0.5 mg/mL protein supplemented with 0.01, 0.05, 0.1, 0.5, or 0.75 mM CPACC, using a similar experimental setup as described above and cumulants analysis.