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Publication Abstract from PubMed

We recently reported the engineering of monomeric streptavidin, mSA, corresponding to a single subunit of wild type (wt) streptavidin tetramer. The monomer was designed by homology modeling, in which the streptavidin and rhizavidin sequences were combined to engineer a high affinity binding pocket containing residues from a single subunit only. Although mSA is stable and binds biotin with nanomolar affinity, its fast off rate (koff ) creates practical challenges during applications. We obtained a 1.9 A crystal structure of mSA bound to biotin to understand their interaction in detail, and used the structure to introduce targeted mutations to improve its binding kinetics. F43 of shwanavidin forms a hydrophobic lid that is important for biotin binding. However, the T48F mutation in mSA, which introduces a comparable hydrophobic lid only results in a modest 20 - 40% improvement in the measured koff . On the other hand, introducing the S25H mutation near the bicyclic ring of bound biotin increases the dissociation half life (t(1/2) ) from 11 min to 83 min at 20 degrees C. Molecular dynamics (MD) simulations suggest that H25 stabilizes the binding loop L3,4 by interacting with A47, and protects key intermolecular hydrogen bonds by limiting solvent entry into the binding pocket. Concurrent T48F or T48W mutation clashes with H25 and partially abrogates the beneficial effects of H25. Taken together, this study suggests that stabilization of the binding loop and solvation of the binding pocket are important determinants of the dissociation kinetics in mSA. (c) Proteins 2013;. (c) 2013 Wiley Periodicals, Inc.

Structure based engineering of streptavidin monomer with a reduced biotin dissociation rate.,Demonte D, Drake EJ, Lim KH, Gulick AM, Park S Proteins. 2013 May 13. doi: 10.1002/prot.24320. PMID:23670729^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.