Figure 6: The C-cap motif of the high-affinity DARPins is disordered. | Scientific Reports

Figure 6: The C-cap motif of the high-affinity DARPins is disordered.

From: Destabilizing an interacting motif strengthens the association of a designed ankyrin repeat protein with tubulin

Figure 6: The C-cap motif of the high-affinity DARPins is disordered.

(a) Gel filtration analysis of 100 μM D1, A-C2 and TM-3. (b) SDS-PAGE analysis of the limited proteolysis of D1, TM-3 and A-C2 by subtilisin. DARPins at 40 μM concentration were incubated at 25 °C with subtilisin at a 1:2000 protease:DARPin molar ratio at the indicated times. (c) Far-UV circular dichroism spectra of 20 μM D1, A-C2 and TM-3 and of 5 μM TM-3 1-149. The spectra were recorded using a 1 mm path length cuvette. They were normalized and are depicted as molar ellipticity. (d) ELISA analysis (tubulin binding and off-rate estimation) of D1, A-C2 and TM-3 along with their C-cap-truncated variants (FL, full length; 1-149, DARPin terminating after residue 149). The experimental conditions were as in Fig. 3d. Here, as in Figs 1 and 3c,d, soluble tubulin at a 100 nM concentration was added after the washing steps to prevent the rebinding of dissociated DARPins.

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