Engineered Streptavidin Monomer and Dimer with Improved Stability and Function Kok Hong Lim, † Heng Huang, ‡ Arnd Pralle, ‡ and Sheldon Park* ,† † Department of Chemical and Biological Engineering and ‡ Department of Physics, University at Buffalo, State University of New York, Buffalo, New York 14260, United States * S Supporting Information 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. S treptavidin and its avian homologue, avidin, bind biotin with high affinity (K d ∼ 10 -15 to 10 -14 M) and are used in molecular detection systems that require stable noncovalent interaction. 1-4 Although they share limited sequence similarity, both molecules adopt a highly conserved tetrameric structure consisting of four identical subunits arranged as two structural dimers. Each of the four subunits in turn forms a common β- barrel motif containing eight antiparallel β-strands. The subunit structure is conserved in all known biotin binding proteins, including the recently identified streptavidin-like molecules from Bradyrhizobium japonicum and Rhizobium etli (proteo- bacterium), Pleurotus cornucopiae (mushroom), and Xenopus tropicalis (frog), which have sequence similarity with streptavidin and avidin in the range of 15-59%. 5-8 The tetrameric architecture is the most common among the streptavidin homologues, suggesting that the tetramer config- uration offers advantages over other possible alternatives. 9 In this regard, biochemical studies have demonstrated that tetramerization plays a structurally and functionally important role in this class of molecules. 10-14 However, rhizavidin binds biotin with high affinity as a dimer, 15 suggesting that tetramerization may not be required for high-affinity biotin binding. The binding pockets of rhizavidin include a disulfide bond between the binding loop 3,4 (L3,4) and strand 6, 15 which may contribute to biotin binding by preorganizing the binding pocket. Designing a functional monomer tests our understanding of the role of oligomerization in streptavidin stability and function and helps extend the reach of streptavidin technology to other novel applications. 16,17 For example, monomeric streptavidin can be used for biotin detection in situations where streptavidin-mediated target aggregation is a concern. In particular, the receptor dimerization is a commonly used mechanism in cell surface signaling, 18 and therefore, labeling cell surface receptors without perturbing cell signaling requires a monovalent reagent. To this end, a monovalent tetramer has been engineered for biophysical studies of the cell receptor dynamics. 19 However, the existing monovalent streptavidin mutants either have limited stability and affinity (monomer) or are cumbersome and wasteful to prepare in vitro (hetero- tetramer). This has motivated us to propose a structure-based solution and engineer new monomers with improved structural and binding properties to add to the existing repertoire of streptavidin mutants. We show that a combination of a disulfide Received: July 5, 2011 Revised: August 25, 2011 Published: September 6, 2011 Article pubs.acs.org/biochemistry © 2011 American Chemical Society 8682 dx.doi.org/10.1021/bi2010366 | Biochemistry 2011, 50, 8682-8691