title: Engineered streptavidin monomer and dimer with improved stability and function.
abstract: Although streptavidin's high affinity for biotin has made it a widely used and studied binding protein and labeling tool, its tetrameric structure may interfere with some assays. A streptavidin mutant with a simpler quaternary structure would demonstrate a molecular-level understanding of its structural organization and lead to the development of a novel molecular reagent. However, modulating the tetrameric structure without disrupting biotin binding has been extremely difficult. In this study, we describe the design of a stable monomer that binds biotin both in vitro and in vivo. To this end, we constructed and characterized monomers containing rationally designed mutations. The mutations improved the stability of the monomer (increase in T(m) from 31 to 47 °C) as well as its affinity (increase in K(d) from 123 to 38 nM). We also used the stability-improved monomer to construct a dimer consisting of two streptavidin subunits that interact across the dimer-dimer interface, which we call the A/D dimer. The biotin binding pocket is conserved between the tetramer and the A/D dimer, and therefore, the dimer is expected to have a significantly higher affinity than the monomer. The affinity of the dimer (K(d) = 17 nM) is higher than that of the monomer but is still many orders of magnitude lower than that of the wild-type tetramer, which suggests there are other factors important for high-affinity biotin binding. We show that the engineered streptavidin monomer and dimer can selectively bind biotinylated targets in vivo by labeling the cells displaying biotinylated receptors. Therefore, the designed mutants may be useful in novel applications as well as in future studies in elucidating the role of oligomerization in streptavidin function.
authors: Department of Chemical and Biological Engineering, University at Buffalo, State University of New York, Buffalo, New York 14260, United States.
authors: Kok Hong
authors: KH
authors: Lim
authors: Heng
authors: H
authors: Huang
authors: Arnd
authors: A
authors: Pralle
authors: Sheldon
authors: S
authors: Park
mesh_terms: D000595:Amino Acid Sequence; D001710:Biotin / Q000378:metabolism; D019281:Dimerization; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D015202:Protein Engineering; D055550:Protein Stability; D019809:Streptavidin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D013302:Streptomyces / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D001710:Biotin; D019809:Streptavidin