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[rightward arrow]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[rightward arrow]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[rightward arrow] 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[rightward arrow]3(N118M). To demonstrate the versatility of the cpAvd4[rightward arrow]3 construct, we have shown that it is possible to link cpAvd4[rightward arrow]3 and cpAvd5[rightward arrow]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.