title: Bifunctional avidin with covalently modifiable ligand binding site.
abstract: The extensive use of avidin and streptavidin in life sciences originates from the extraordinary tight biotin-binding affinity of these tetrameric proteins. Numerous studies have been performed to modify the biotin-binding affinity of (strept)avidin to improve the existing applications. Even so, (strept)avidin greatly favours its natural ligand, biotin. Here we engineered the biotin-binding pocket of avidin with a single point mutation S16C and thus introduced a chemically active thiol group, which could be covalently coupled with thiol-reactive molecules. This approach was applied to the previously reported bivalent dual chain avidin by modifying one binding site while preserving the other one intact. Maleimide was then coupled to the modified binding site resulting in a decrease in biotin affinity. Furthermore, we showed that this thiol could be covalently coupled to other maleimide derivatives, for instance fluorescent labels, allowing intratetrameric FRET. The bifunctional avidins described here provide improved and novel tools for applications such as the biofunctionalization of surfaces.
authors.affiliation: Institute of Medical Technology, University of Tampere and Tampere University Hospital, Tampere, Finland.
authors.forename: Jenni
authors.initials: J
authors.lastname: Leppiniemi
authors.forename: Juha A E
authors.initials: JA
authors.lastname: Määttä
authors.forename: Henrik
authors.initials: H
authors.lastname: Hammaren
authors.forename: Mikko
authors.initials: M
authors.lastname: Soikkeli
authors.forename: Mikko
authors.initials: M
authors.lastname: Laitaoja
authors.forename: Janne
authors.initials: J
authors.lastname: Jänis
authors.forename: Markku S
authors.initials: MS
authors.lastname: Kulomaa
authors.forename: Vesa P
authors.initials: VP
authors.lastname: Hytönen
mesh_terms: D001360:Avidin / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites / Q000235:genetics*; D001710:Biotin / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000138:chemical synthesis*; D005818:Genetic Engineering / Q000379:methods; D008024:Ligands; D017354:Point Mutation; D013438:Sulfhydryl Compounds / Q000737:chemistry
chemical_list: D003432:Cross-Linking Reagents; D008024:Ligands; D013438:Sulfhydryl Compounds; D001360:Avidin; D001710:Biotin