Binding of phenylphosphocholine-carrier conjugates to the combining site of antibodies maintains a conformation of the hapten.
The structural basis of the binding of phenylphosphocholine haptens to antibodies was studied. This was done by preparing antibodies and testing binding to conjugates of phenylphosphocholine. The choice of haptens was made in order to evaluate the contribution of the carrier to binding, and its effect on hapten conformation in the active site. Thus, phosphocholine (PC) was diazophenyl-linked to tyrosine or histidine as single amino acid carriers and to tripeptides or octapeptides containing tyrosine or histidine as central amino acids to which PC was attached. Relative affinity was assessed by inhibition enzyme-linked immunosorbent assay (ELISA) and binding constants were determined by fluorescence quenching. Fluorinated haptens were used to determine the kinetics of binding using 19F nuclear magnetic resonance. The transferred nuclear Overhauser effect was used to characterize conformation of the bound hapten. We had previously shown that nitrophenylphosphocholine unlinked to carrier is bound in the active site as a bent structure [Bruderer, U., Peyton, D. H., Barbar, E., Fellman, J. H., & Rittenberg, M. B. (1992) Biochemistry 31, 584-589]. We show here that this same bent conformation is retained in the active site regardless of the neighboring carrier or the conformation of the hapten in the unbound conjugate. The presence of the carrier residues in the bound state does, however, influence affinity.
Barbar
E
E
Department of Chemistry, Portland State University, Oregon 97207-0751, USA.
Martin
T M
TM
Brown
M
M
Rittenberg
M B
MB
Peyton
D H
DH
D000595:Amino Acid Sequence; D000906:Antibodies; D001666:Binding Sites, Antibody; D017931:DNA Primers; D006241:Haptens; D007128:Immunoglobulin Fragments / Q000096:biosynthesis / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009596:Nitrophenols / Q000138:chemical synthesis; D009943:Organophosphorus Compounds; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D013329:Structure-Activity Relationship; D013816:Thermodynamics
D000906:Antibodies; D017931:DNA Primers; D006241:Haptens; D007128:Immunoglobulin Fragments; D009596:Nitrophenols; D009943:Organophosphorus Compounds; D011994:Recombinant Proteins; C008644:nitrophenylphosphate