Rational Modification of Ligand‐Binding Preference of Avidin by Circular Permutation and Mutagenesis

Chicken avidin is a key component used in a wide variety of biotechnological applications. Here we present a circularly permuted avidin (cpAvd4→3) that lacks the loop between β‐strands 3 and 4. Importantly, the deletion of the loop has a positive effect on the binding of 4′‐hydroxyazobenzene‐2‐carboxylic acid (HABA) to avidin. To increase the HABA affinity of cpAvd4→3 even further, we mutated asparagine 118 on the bottom of the ligand‐binding pocket to methionine, which simultaneously caused a significant drop in biotin‐binding affinity. The X‐ray structure of cpAvd4→ 3(N118M) allows an understanding of the effect of mutation to biotin‐binding, whereas isothermal titration calorimetry revealed that the relative binding affinity of biotin and HABA had changed by over one billion‐fold between wild‐type avidin and cpAvd4→3(N118M). To demonstrate the versatility of the cpAvd4→3 construct, we have shown that it is possible to link cpAvd4→3 and cpAvd5→4 to form the dual‐chain avidin called dcAvd2. These novel avidins might serve as a basis for the further development of self‐organising nanoscale avidin building blocks.