MUTATIONAL ANALYSIS OF ACTIVE-SITE CONTACT RESIDUES IN ANTIFLUORESCEIN MONOCLONAL-ANTIBODY 4-4-20

The contribution to high affinity F1 binding by each crystallographica lly defined Mab 4-4-20 (K-alpha= 1.7 x 10(10) M(-1); Q(max) = 90%) lig and contact residue (L27d(His), L32(Tyr), L34(Arg) L91(Ser): L96(Trp) and H33(Trp)) has been determined by site-specific mutagenesis studies . All six antigen contact residues were changed to Ala in the single-c hain derivative of Mab 4-4-20 and following expression in E. coli, den aturation, refolding and purification, each SCA mutant was characteriz ed in terms of F1 binding affinity, Q(max) lambda(max) and idiotype. R esults demonstrated that Ala substitutions at each ligand contact resi due reduced the binding affinities and quenching maxima for an residue s except L27d which retained wild type characteristics. The SCA (Tyr)L 32(Ala), (Ser)L(91Ala) and (Trp)H33(Ala) mutants exhibited binding aff inities that were similar to 1000-fold lower than the wild type value and greatly reduced Q(max) values. Additionally, other amino acid subs titutions were performed at three of the six antigen contact residues (L91(Ser), L96(Trp) and H33(Trp)) to further evaluate the role of each in F1 binding. Therefore, the following mutations were constructed an d characterized: (Ser)L91(Asn), (Trp)L96(Tyr), (Trp)L96(Phe), (Trp)L96 (Leu), (Trp)H33(Tyr) and (Trp)PH33(Phe). Results of site-specific muta genesis studies are discussed in terms of Mab active site structure an d suggest that L32(Tyr), L91(Ser) and H33(Trp) are important for high affinity F1 binding and efficient F1 quenching.