期刊
JOURNAL OF MOLECULAR BIOLOGY
卷 426, 期 1, 页码 199-214出版社
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2013.09.016
关键词
avidin; protein design; bivalent; supramolecular; nanotechnology
资金
- Biotechnology and Biological Sciences Research Council (BBSRC)
- Wellcome Trust
- Department of Biochemistry, University of Oxford
- Biotechnology and Biological Sciences Research Council [BB/I006303/1] Funding Source: researchfish
- BBSRC [BB/I006303/1] Funding Source: UKRI
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 1.4 angstrom resolution and trans-divalent streptavidin to 1.6 angstrom 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. (C) 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
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