Figure 3.

Activity-stability trade-off in single and 2nd generation methyltransferase mutants. (a) Grouping mutants as a function of folded-states, stability and transferase activity. The top right blue panel contains variants with WT-like stability and activity. The lower panels contain mutants with T_m1 > 5 °C lower than WT. The mutants in the activity penalty panels but above the ‘activity assay detection limit’ are variants with transferase activity lower than 60% of WT activity. The variants in the lower left red panel appeared as misfolded in the analysis of their tryptophan fluorescence barycentric mean shift upon unfolding (Supplementary Fig. 4). (b) Activity ratios (Fig. 2) plotted against the first transition point (T_m1) of the melting curves. WT is in black, single mutants are in light blue, single A201 mutants are in purple, double mutants are in dark blue, E46Q mutants in brown and triple mutants in green. (c) Negative epistasis between second and third shell residues. ΔT_m is the variation between the first transition points in the melting curves of WT and mutant. The expected ΔT_m corresponds to the ΔT_m value if the effects of mutations are purely additive. Any deviation from the diagonal line suggests epistatic interactions between the mutations. All T_m and ΔT_m values are reported in the Supplementary Table 1.