Rational design of an active avidin monomer.
Homotetrameric chicken avidin that binds four molecules of biotin was converted to a monomeric form (monoavidin) by mutations of two interface residues: tryptophan 110 in the 1 --> 2 interface was mutated to lysine and asparagine 54 in the 1 --> 4 interface was converted to alanine. The affinity for biotin binding of the mutant decreased from K(d) approximately 10(-15) m of the wild-type tetramer to K(d) approximately 10(-7) m, which was studied by an optical biosensor IAsys and by a fluorescence spectroscopical method in solution. The binding was completely reversible. Conversion of the tetramer to a monomer results in increased sensitivity to proteinase K digestion. The antigenic properties of the mutated protein were changed, such that monoavidin was only partially recognized by a polyclonal antibody whereas two different monoclonal antibodies entirely failed to recognize the avidin monomer. This new monomeric avidin, which binds biotin reversibly, may be useful for applications both in vitro and in vivo. It may also shed light on the effect of intersubunit interactions on the binding of ligands.
Laitinen
Olli H
OH
Department of Biological and Environmental Science, P. O. Box 35, FIN-40014 University of Jyväskylä, Finland.
Nordlund
Henri R
HR
Hytönen
Vesa P
VP
Uotila
Sanna T H
ST
Marttila
Ari T
AT
Savolainen
Janne
J
Airenne
Kari J
KJ
Livnah
Oded
O
Bayer
Edward A
EA
Wilchek
Meir
M
Kulomaa
Markku S
MS
D000818:Animals; D001360:Avidin / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics; D001704:Biopolymers; D001710:Biotin / Q000378:metabolism; D002645:Chickens; D002850:Chromatography, Gel; D004797:Enzyme-Linked Immunosorbent Assay; D008958:Models, Molecular; D016296:Mutagenesis; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D001704:Biopolymers; D011994:Recombinant Proteins; D001360:Avidin; D001710:Biotin