Figure 3: Effect of mutations on the thermostability of APC1.
(a) Effect of mutations on the apparent thermostability of APC1. The apparent melting temperatures of 28 alanine replacement mutants were determined by using the CPM thermostability assay. Either 2.5 mM calcium chloride (light gray bars) or EGTA (dark gray bars) were added to the sample 10 minutes before temperature ramping. (b) The difference in Tm of each mutant from the Tm of wild-type APC1 was calculated. For each mutant, the ∆Tm in the calcium condition was plotted against the ∆Tm in the EGTA condition. Mutations that have a similar effect on stability to one another in both the presence and absence of calcium cluster together on the graph, and have been grouped accordingly with a colored line. Mutations on the diagonal line (black dashed line) have a proportional effect on stability in the presence and absence of calcium. Mutations on the parallel line (red dashed line), shifted by 9 °C relative to the wild-type, have a disproportional effect on stability in one condition over the other, indicating an effect on regulation.
