Introduction of histidine residues into avidin subunit interfaces allows pH-dependent regulation of quaternary structure and biotin binding |
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Authors: | Nordlund Henri R Hytönen Vesa P Laitinen Olli H Uotila Sanna T H Niskanen Einari A Savolainen Janne Porkka Eevaleena Kulomaa Markku S |
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Institution: | Department of Biological and Environmental Science, P.O. Box 35, FIN-40014 University of, Jyv?skyl?, Finland. |
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Abstract: | In order to turn the subunit association and biotin binding of avidin into pH-sensitive phenomena, we have replaced individually three amino acid residues in avidin (Met96, Val115 and Ile117) with histidines in the 1-3 interface, and in combination with a histidine conversion in the 1-2 interface (Trp110). The single replacements Met96His and Val115His in the 1-3 interface were found to have a clear effect on the quaternary structure of avidin, since subunit associations of these mutants became pH-dependent. The histidine replacement in the 1-2 interface affected the biotin-binding properties of the mutants, in particular reversibility of binding and protein-ligand complex formation were pH-sensitive, as measured by IAsys biosensor and fluorescence correlation spectroscopy, respectively. The possibility of regulating the quaternary structure and function of avidin in a controlled and predictable manner, due to introduced interface histidines, will expand even further the range and versatility of the avidin-biotin technology. |
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Keywords: | Avidin pH dependence Protein engineering |
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