Structural consequences of cutting a binding loop: two circularly permuted variants of streptavidin
- University of Washington, Box 357420, Seattle, WA 98195-7420 (United States)
- University of Washington, Box 355061, Seattle, WA 98195-5061 (United States)
- Vanderbilt University, 5142 Medical Research Building III, 465 21st Avenue South, Nashville, TN 37232-8725 (United States)
The crystal structures of two circularly permuted streptavidins probe the role of a flexible loop in the tight binding of biotin. Molecular-dynamics calculations for one of the mutants suggests that increased fluctuations in a hydrogen bond between the protein and biotin are associated with cleavage of the binding loop. Circular permutation of streptavidin was carried out in order to investigate the role of a main-chain amide in stabilizing the high-affinity complex of the protein and biotin. Mutant proteins CP49/48 and CP50/49 were constructed to place new N-termini at residues 49 and 50 in a flexible loop involved in stabilizing the biotin complex. Crystal structures of the two mutants show that half of each loop closes over the binding site, as observed in wild-type streptavidin, while the other half adopts the open conformation found in the unliganded state. The structures are consistent with kinetic and thermodynamic data and indicate that the loop plays a role in enthalpic stabilization of the bound state via the Asn49 amide–biotin hydrogen bond. In wild-type streptavidin, the entropic penalties of immobilizing a flexible portion of the protein to enhance binding are kept to a manageable level by using a contiguous loop of medium length (six residues) which is already constrained by its anchorage to strands of the β-barrel protein. A molecular-dynamics simulation for CP50/49 shows that cleavage of the binding loop results in increased structural fluctuations for Ser45 and that these fluctuations destabilize the streptavidin–biotin complex.
- OSTI ID:
- 22351288
- Journal Information:
- Acta Crystallographica. Section D: Biological Crystallography, Vol. 69, Issue Pt 6; Other Information: PMCID: PMC3663120; PMID: 23695241; PUBLISHER-ID: be5218; OAI: oai:pubmedcentral.nih.gov:3663120; Copyright (c) International Union of Crystallography 2013; Country of input: International Atomic Energy Agency (IAEA); ISSN 0907-4449
- Country of Publication:
- Denmark
- Language:
- English
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