Fig. 2: Luciferase disaggregation and refolding and ATPase activity measurements of CLPB; Structural analysis of the pathogenic variants using a homology model of CLPB.

a Left: In vitro luciferase disaggregation and refolding by purified wild type CLPB (WT-CLPB) protein and its variants. Chemically denatured luciferase was added to reaction mixtures containing 0.5 µM CLPB WT protein or indicated variants in the presence of 5 mM ATP. Aliquots were taken at indicated timepoints and the luciferase activity was assayed. The CLPB disaggregase and refolding activity is shown by the gradual return of luciferase activity with time during the incubation. Note the completely absent disaggregase and refolding activity in two of the mutants and some residual activity in the Lys404Thr mutant. Inset: curves for low activity variants, rescaled for clarity. Right: ATPase activity of CLPB WT and variants. b Luciferase disaggregation and refolding activity of 1:1 mixtures of WT-CLPB with each of the pathogenic CLPB variants. The WT-CLPB reaction contains 0.25 µM CLPB and 0.5 µM bovine serum albumin while reactions with indicated pathogenic variants contain both 0.25 µM of WT-CLPB and 0.25 µM of pathogenic variant (total 0.5 µM CLPB protein). The luciferase activity was measured after a 4-hour incubation period to allow reactivation. c Molecular gel filtration of WT-CLPB and 1:1 mixtures of WT and indicated variants, separated on Superose 6 Increase 10/300. Positions of molecular weight markers indicated. d Structural analysis of the pathogenic variants using a homology model of CLPB. Overview of the model of the CLPB C-terminal domain showing two neighboring monomers. Two subunits are shown in different shades of gray. The ADP molecule is shown in ball representation (yellow). The positions of the pathogenic variants, one subunit only, are shown in red (note that Glycine does not have a sidechain). Sidechain atoms are shown in ball representation where possible. e Details of pathogenic variants G560R and R561W. Sidechain atoms are shown in stick representation to show the differences between wild type (green) and mutant (red). (Glycine sidechain not visible.) f Details of pathogenic variant K404T. g Details of pathogenic variant P427L.