Clinical mutations in the ankyrin binding region of β-spectrin disrupt protein stability rather than binding affinity. (A) The characterized human β-spectrin mutants can be classified into 4 categories: those from clinical³⁸  sources or functionally based³⁹  experiments (orange), those along the helix A of repeat 14 interface (green), those along helix C of repeat 14 (blue), and those in the B/C loop of repeat 15 (purple). The ankyrin mutations coloring is assigned on the basis of the position of their spectrin binding partner where applicable. (B) Surface plasmon resonance measurements of the binding affinity (color-coded as in panel A) illustrate that structure-guided mutations disrupt binding significantly, whereas many clinical mutations do not. The inset depicts the fit of each mutant's sensorgram upon injection of ZU5-ANK at 200nM. (C) In contrast, a similar analysis of thermal stability demonstrated that clinical mutations have a significant destabilizing effect, whereas structure-based mutations do not. Bars indicate average values with error bars corresponding to the SE. The insert presents the circular dichroism thermal denaturation data (fraction unfolded vs temperature) and 2-state fits for each mutant.