A streptavidin mutant with altered ligand-binding specificity.
The biotin-binding site of streptavidin was modified to alter its ligand-binding specificity. In natural streptavidin, the side chains of N23 and S27 make two of the three hydrogen bonds with the ureido oxygen of biotin. These two residues were mutated to severely weaken biotin binding while attempting to maintain the affinity for two biotin analogs, 2-iminobiotin and diaminobiotin. Redesigning of the biotin-binding site used the difference in local electrostatic charge distribution between biotin and these biotin analogs. Free energy calculations predicted that the introduction of a negative charge at the position of S27 plus the mutation N23A should disrupt two of the three hydrogen bonds between natural streptavidin and the ureido oxygen of biotin. In contrast, the imino hydrogen of 2-iminobiotin should form a hydrogen bond with the side chain of an acidic amino acid at position 27. This should reduce the biotin-binding affinity by approximately eight orders of magnitude, while leaving the affinities for these biotin analogs virtually unaffected. In good agreement with these predictions, a streptavidin mutant with the N23A and S27D substitutions binds 2-iminobiotin with an affinity (Ka) of 1 x 10(6) M-1, two orders of magnitude higher than that for biotin (1 x 10(4) M-1). In contrast, the binding affinity of this streptavidin mutant for diaminobiotin (2.7 x 10(4) M-1) was lower than predicted (2.9 x 10(5) M-1), suggesting the position of the diaminobiotin in the biotin-binding site was not accurately determined by modeling.
Reznik
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Center for Advanced Biotechnology and Departments of Physics, Biomedical Engineering, and Pharmacology and Experimental Therapeutics, Boston University, Boston, MA 02215, USA.
Vajda
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Sano
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Cantor
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D001665:Binding Sites / Q000235:genetics; D001710:Biotin / Q000737:chemistry; D015195:Drug Design; D004926:Escherichia coli; D008024:Ligands; D009154:Mutation; D011487:Protein Conformation; D019809:Streptavidin / Q000737:chemistry* / Q000235:genetics*
D008024:Ligands; D001710:Biotin; D019809:Streptavidin