Figure 4.

Modifications and randomizations at the capping repeats and surface entropy reduction of the DARPin backside. DARPins of the original (off7, left, PDB 1SVX) and new (C7_16, right, PDB 4JB8) library are shown in cartoon representation with diversified/modified residues highlighted as colored sticks or in surface representation. (A and C) Bulky E20 and R23 residues of the N-cap are frequently involved in target contacts [Fig. 2(A)] and were exchanged for the smaller D20 and S23 in the new library. The positions 1–3 of the C-cap (β-turn) were subjected to randomization in the new library following the same scheme as depicted in Figure 1(C). (B) DARPins viewed from their backside. By mutating E21D and K25A in each internal repeat [Fig. 1(C)], the surface entropy of the DARPin backside was reduced in the new library, which is expected to facilitate crystallization. (C) Sequences of the N- and C-caps are shown and modifications of the original library are highlighted.