Figure 3.

Using IMPROvER to identify stabilising variants of ClPPase. (A) Comparison of residues selected by IMPROvER modules but excluded due to prior known critical role. (B) Comparison of residues selected by IMPROvER that are overlapping between the different modules. (C) Statistical analysis of all data collected for ClPPase variant in-gel GFP-based stability assay, or grouped by prediction module. Each data point represents a biological repeat of the single-temperature stability analysis (single-temperature challenge at 50 ${}^{\circ }$C normalised to intensity of sample incubated on ice). In each case the median of all points is displayed with 95% confidence intervals represented as whiskers. (D) wild-type (WT) ClPPase, and representative variant curves of three stabilisers and one destabiliser, assayed by ten-temperature stability analysis (panel insert and Fig. S4) after temperature challenge at 20, 30, 40, 45, 50, 55, 60, 70, 80, and 90 ${}^{\circ }$C. Data in each curve are normalised to the intensity of sample incubated on ice. The wild-type curve was collected as an average of eight biological repeats and variants were three biological repeats. Please see Fig. S2A for an uncropped gel image. (E) $T_m$ of top stabilising variants (and the destabilising variant L142P) versus their specific ${\text{PP}}_{\mathrm{i}}$ hydrolysis activity. Error bars in all panels D and E are representative of SEM. Stabilising variants that retain or improve on wild-type activity cluster in the top right quadrant. (F) View in the plane of the membrane and (G) perpendicular to the membrane at the most stabilising variant positions (coloured balls) mapped to the best comparative homology model of ClPPase.