Biotin Induces Tetramerization of a Recombinant Monomeric Avidin

Abstract

Chicken avidin, a homotetramer that binds four molecules of biotin was converted to a monomeric form by successive mutations of interface residues to alanine. The major contribution to monomer formation was the mutation of two aspartic acid residues, which together account for ten hydrogen bonding interactions at the 1-4 interface. Mutation of these residues, together with the three hydrophobic residues at the 1-3 interface, led to stable monomer formation in the absence of biotin. Upon addition of biotin, the monomeric avidin reassociated to the tetramer, which exhibited properties similar to those of native avidin, with respect to biotin binding, thermostability, and protease resistance. To our knowledge, these unexpected results represent the first example of a small monovalent ligand that induces oligomerization of a monomeric protein. This study may suggest a biological role for low molecular weight ligands in inducing oligomerization and in maintaining the stability of multimeric protein assemblies.

Engineered single-chain dimeric streptavidins with an unexpected strong preference for biotin-4-fluorescein

“…These results show that binding of biotin or B4F favors tetramer formation, presumably because of the stabilization afforded by the interaction between W120 of the adjacent dimer with the bound ligand. This is consistent with other studies showing that ligand binding favors streptavidin oligomerization (10,(24)(25)(26).…”

Multiphoton fabrication of chemically responsive protein hydrogels for microactuation

“…These results are consistent with the well characterized stabilizing effect of biotin on avidin (35,38) and show the possibility for achieving microstructure actuation by means of biotin binding. By using engineered avidins (37,39), biotininduced actuation can be explored under a wider range of environmental conditions.…”

Targeting of biotinylated compounds to its target tissue using a low-density lipoprotein receptor–avidin fusion protein

“…Tetramerization of the fusion protein might be a stabilizing factor, as it is with avidin. 27 The oligomerization state of the fusion protein is of interest because it may greatly influence the biotin-binding affinity. For tetrameric avidin, the K d is B10 À15 (M), whereas for the monomeric avidin the affinity is greatly reduced (K d B10 À7 -10 À8 M).…”