Introduction of histidine residues into avidin subunit interfaces allows pH-dependent regulation of quaternary structure and biotin binding.
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.
Nordlund
Henri R
HR
Department of Biological and Environmental Science, P.O. Box 35, FIN-40014 University of, Jyväskylä, Finland.
Hytönen
Vesa P
VP
Laitinen
Olli H
OH
Uotila
Sanna T H
ST
Niskanen
Einari A
EA
Savolainen
Janne
J
Porkka
Eevaleena
E
Kulomaa
Markku S
MS
D019943:Amino Acid Substitution; D000818:Animals; D001360:Avidin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods; D001710:Biotin / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007313:Insecta; D008958:Models, Molecular; D008970:Molecular Weight; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods
D021122:Protein Subunits; D011994:Recombinant Proteins; D001360:Avidin; D006639:Histidine; D001710:Biotin