Evolved streptavidin mutants reveal key role of loop residue in high-affinity binding

Maria L B Magalhães, Clarissa Melo Czekster, Rong Guan, Vladimir N. Malashkevich, Steven C. Almo, Matthew Levy

Research output: Contribution to journalArticle

15 Citations (Scopus)

Abstract

We have performed a detailed analysis of streptavidin variants with altered specificity towards desthiobiotin. In addition to changes in key residues which widen the ligand binding pocket and accommodate the more structurally flexible desthiobiotin, the data revealed the role of a key, non-active site mutation at the base of the flexible loop (S52G) which slows dissociation of this ligand by approximately sevenfold. Our data suggest that this mutation results in the loss of a stabilizing contact which keeps this loop open and accessible in the absence of ligand. When this mutation was introduced into the wild-type protein, destabilization of the opened loop conferred a ∼10-fold decrease in both the on-rate and off-rate for the ligand biotin-4-fluoroscein. A similar effect was observed when this mutation was added to a monomeric form of this protein. Our results provide key insight into the role of the streptavidin flexible loop in ligand binding and maintaining high affinity interactions. Published by Wiley-Blackwell.

Original languageEnglish (US)
Pages (from-to)1145-1154
Number of pages10
JournalProtein Science
Volume20
Issue number7
DOIs
StatePublished - Jul 2011

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Streptavidin
Ligands
Mutation
Biotin
Proteins

Keywords

  • Dissociation kinetics
  • In vitro compartmentalization
  • Loop
  • Streptavidin

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Magalhães, M. L. B., Czekster, C. M., Guan, R., Malashkevich, V. N., Almo, S. C., & Levy, M. (2011). Evolved streptavidin mutants reveal key role of loop residue in high-affinity binding. Protein Science, 20(7), 1145-1154. https://doi.org/10.1002/pro.642

Evolved streptavidin mutants reveal key role of loop residue in high-affinity binding. / Magalhães, Maria L B; Czekster, Clarissa Melo; Guan, Rong; Malashkevich, Vladimir N.; Almo, Steven C.; Levy, Matthew.

In: Protein Science, Vol. 20, No. 7, 07.2011, p. 1145-1154.

Research output: Contribution to journalArticle

Magalhães, MLB, Czekster, CM, Guan, R, Malashkevich, VN, Almo, SC & Levy, M 2011, 'Evolved streptavidin mutants reveal key role of loop residue in high-affinity binding', Protein Science, vol. 20, no. 7, pp. 1145-1154. https://doi.org/10.1002/pro.642
Magalhães, Maria L B ; Czekster, Clarissa Melo ; Guan, Rong ; Malashkevich, Vladimir N. ; Almo, Steven C. ; Levy, Matthew. / Evolved streptavidin mutants reveal key role of loop residue in high-affinity binding. In: Protein Science. 2011 ; Vol. 20, No. 7. pp. 1145-1154.
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