Figure 4

Activities of variants designed using substitution weights. Activities towards N-Succinyl-Ala-Ala-Pro-Leu p-nitroanilide were measured at 37°C following a 5 minute heat treatment of the enzyme at 68°C. Activities are expressed relative to the mean activity of duplicates of wild-type proteinase K. Error bars represent one standard deviation of the activity measurements. Variants are grouped according to the machine learning algorithm used to calculate substitution weights (indicated below each group), and are compared with the best variants from the initial design set (variants 1–40 and 1–50 black bars, on the left). The first design (yellow bars, design method G in Additional file 2) of each group belongs to set 2. We included a substitution in the design if it occurred at least three times in the training set and its mean weight was at least one standard deviation above zero. All remaining designs in each group belong to set 3. The second in each group (green bars, design method J in Additional file 2) includes substitutions occurring at least three times and whose mean weights were merely positive (eg Figure 3A, red and blue circles). The third in each group (red bars, design method K in Additional file 2) contained all substitutions occurring at least three times and whose mean weight was at least one standard deviation above zero (eg Figure 3A, red circles). Note that this third design in each group is always better than the second. The last variant(s) in each group (blue bars, design method L in Additional file 2) were designed by modeling interdependent substitutions (eg Figure 3B, red circles).