Regulation of the flavin redox potential by flavin-binding antibodies.
Single-chain Fv antibody fragments binding different flavin forms [10-(5'-carboxybutyl-)flavin (Fl[ox]) and 10-(5'-carboxybutyl)-1,5-dihydroflavin (Fl[red])] have been generated from an antibody phage-display library to study how a protein environment regulates the redox potential, starting from a protein other than a natural flavoprotein. These 'flavobodies' are characterized by time-resolved and steady-state fluorescence spectroscopy, by competitive ELISA methods (mapping of the antigen-binding site), and by molecular modelling. The three-dimensional models of the antigen-binding sites are consistent with the experimental results. Binding of anti-Fl(red) 5 to flavin increases the redox potential, mainly due to an Arg residue interacting with the flavin N1. Thus anti-Fl(red) 5 shows an 'oxidase-like' redox-potential behaviour, confirming the idea that positively charged residues in the vicinity of N1 increase the redox potential. The results obtained with anti-Fl(ox), which do not resemble a natural flavoprotein, show that when the pyrimidine-like nucleus of the flavin is not involved in binding, the redox potential is not significantly affected. These results are in contrast to those obtained with chicken riboflavin-binding protein.
Bruggeman
Y E
YE
Laboratory for Biochemistry, Agricultural University Wageningen, The Netherlands.
Honegger
A
A
Kreuwel
H
H
Visser
A J
AJ
Laane
C
C
Schots
A
A
Hilhorst
R
R
D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry*; D001666:Binding Sites, Antibody; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D005415:Flavins / Q000737:chemistry* / Q000276:immunology*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry; D007147:Immunoglobulin Light Chains / Q000737:chemistry; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D019151:Peptide Library; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D000906:Antibodies; D005415:Flavins; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D019151:Peptide Library; D011994:Recombinant Proteins