Figure 3: Assessing the contribution of the D1 residues mutated in A-C2.

(a) Sequence alignment of D1, A-C2 and the optimized TM-3 DARPin (color code: N-cap, green; 1^st internal repeat, bright blue, 2^nd internal repeat, dark blue; 3^rd internal repeat, cyan; C-cap, yellow; mutated residues relative to D1, red). (b) Structure of tubulin−D1⁹ with the Cα position of the residues mutated in A-C2 highlighted as red spheres. D1 is colored according to panel A. (c) ELISA analysis (binding and off-rate estimation) of the 11 D1 single mutants, 2 double mutants (DM-1: H118R, I122V; DM-2: E127K, V131A) and of 2 triple mutants (TM-1: N74S, I76S, I78L; TM-2: F150Y, I152T, N158S). The experimental conditions were as in Fig. 1. (d) ELISA analysis of the DM-3 double mutant (I152T, N158S) and of the TM-3 triple mutant (with the additional H118R substitution) together with the corresponding single mutants. The experimental conditions were as in panel C, except that the concentration of biotinylated peptide-coupled tubulin used for coating was decreased 5-fold (down to 2 nM).