Dual‐chain and single‐chain avidins: From subunits to protein domains via circularly permuted avidins

Avidin (Avd), a chicken tetrameric biotin‐binding protein, has been extensively engineered by rational mutagenesis. Since the mutated Avds are single gene products, the desired changes took effect in all subunits. Avd molecules were constructed here with 4 binding sites first in 2 and then in 1 polypeptide chain. The native N‐ and C‐termini were connected by a linker and the new termini introduced into 2 different loops. These circularly permuted Avds (cpAvd) were fused by another linker to form a pseudo‐tetrameric dual‐chain Avd (dcAvd). Two dcAvds formed a dimer with biotin‐binding and structural characteristics similar to those of wt Avd. This scaffold was then used to generate dual‐affinity Avds (daAvd) where the neighbouring biotin‐binding sites had different affinities for biotin. Single chain Avd (scAvd) was constructed by fusion of 2 dcAvds. It contains 4 cpAvd domains with short linkers connecting them into a single polypeptide chain. ScAvd showed biotin binding and thermal stability similar to wt Avd. In contrast to wt Avd, scAvd enables each of the 4 domains to be independently modified. In conclusion, the dcAvd and scAvd scaffold can be used to construct spatially and stoichiometrically defined pseudo‐tetrameric Avds with altered domain characteristics. This subunit‐to‐domain strategy can also be applied to other proteins and protein complexes in new applications in life sciences and nanotechnology.