Molecular recognition in the streptavidin-biotin system

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Molecular recognition in the streptavidin-biotin system

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Title: Molecular recognition in the streptavidin-biotin system
Author: Chu, Vano
Abstract: In this project, the bases for high-affinity interaction and ligand recognition were investigated in the streptavidin-biotin system. With an estimated binding constant on the order of 1013 M-1, the binding of biotin to streptavidin is one of the strongest noncovalent interactions known. Here, we report the results of combined structural and thermodynamic efforts toward defining the contributions of two components of the tight-binding phenomenon---a flexible loop which closes over the binding site and a key residue in the hydrogen bond network to biotin. To probe the role of the loop, a new mutagenesis technique based on creating circularly permuted variants of core streptavidin was used to create discontinuities in the binding-site loop and to remove it entirely through relocation of the termini without changing the overall structure of the protein. The loop alterations result in drastic reductions in binding affinity accompanied by large increases in the dissociation rate of biotin from the binding site. The results of the flexible loop alterations underscore the magnitude of the contribution which loop closure provides to the stability of the streptavidin-biotin complex. Also, site-directed mutagenesis was used to remove a hydrogen bond from aspartate-128 to a ureido nitrogen of biotin by changing the residue to alanine (D128A). This modification also generated a large drop in affinity with a concomitant increase in dissociation rates. Parallel studies in x-ray crystallography and in molecular modeling provided the surprising finding that the structure of the D128A-biotin complex very closely resembles an intermediate structure seen in most of the computed dissociation pathways. The D128A-biotin complex may be viewed essentially as a stable, structural snapshot of an intermediate state on the dissociation reaction coordinate.
Description: Thesis (Ph. D.)--University of Washington, 1998
URI: http://hdl.handle.net/1773/8106

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