Plug-and-play pairing via defined divalent streptavidins.
Streptavidin is one of the most important hubs for molecular biology, either multimerizing biomolecules, bridging one molecule to another, or anchoring to a biotinylated surface/nanoparticle. Streptavidin has the advantage of rapid ultra-stable binding to biotin. However, the ability of streptavidin to bind four biotinylated molecules in a heterogeneous manner is often limiting. Here, we present an efficient approach to isolate streptavidin tetramers with two biotin-binding sites in a precise arrangement, cis or trans. We genetically modified specific subunits with negatively charged tags, refolded a mixture of monomers, and used ion-exchange chromatography to resolve tetramers according to the number and orientation of tags. We solved the crystal structures of cis-divalent streptavidin to 1.4Å resolution and trans-divalent streptavidin to 1.6Å resolution, validating the isolation strategy and explaining the behavior of the Dead streptavidin variant. cis- and trans-divalent streptavidins retained tetravalent streptavidin's high thermostability and low off-rate. These defined divalent streptavidins enabled us to uncover how streptavidin binding depends on the nature of the biotin ligand. Biotinylated DNA showed strong negative cooperativity of binding to cis-divalent but not trans-divalent streptavidin. A small biotinylated protein bound readily to cis and trans binding sites. We also solved the structure of trans-divalent streptavidin bound to biotin-4-fluorescein, showing how one ligand obstructs binding to an adjacent biotin-binding site. Using a hexaglutamate tag proved a more powerful way to isolate monovalent streptavidin, for ultra-stable labeling without undesired clustering. These forms of streptavidin allow this key hub to be used with a new level of precision, for homogeneous molecular assembly.
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
Michael
M
Fairhead
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
Denis
D
Krndija
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK.
Ed D
ED
Lowe
Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. Electronic address: mark.howarth@bioch.ox.ac.uk.
Mark
M
Howarth
D001710:Biotin / Q000378:metabolism*; D018360:Crystallography, X-Ray; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019809:Streptavidin / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D011994:Recombinant Proteins; D001710:Biotin; D019809:Streptavidin
2-methyl-2,4-pentanediol; LDLR; MPD; PBS; PDB; Protein Data Bank; avidin; bivalent; low-density lipoprotein receptor; nanotechnology; phosphate-buffered saline; protein design; supramolecular