Mutational analysis of active site contact residues in anti-fluorescein monoclonal antibody 4-4-20.
The contribution to high affinity Fl binding by each crystallographically defined Mab 4-4-20 (Ka = 1.7 x 10(10) M-1; Qmax = 90%) ligand contact residue (L27dHis, L32Tyr, L34Arg, L91Ser, L96Trp and H33Trp) has been determined by site-specific mutagenesis studies. All six antigen contact residues were changed to Ala in the single-chain derivative of Mab 4-4-20 and following expression in E. coli, denaturation, refolding and purification, each SCA mutant was characterized in terms of Fl binding affinity, Qmax, lambda max and idiotype. Results demonstrated that Ala substitutions at each ligand contact residue reduced the binding affinities and quenching maxima for all residues except L27d which retained wild type characteristics. The SCA TyrL32Ala, SerL91Ala and TrpH33Ala mutants exhibited binding affinities that were approximately 1000-fold lower than the wild type value and greatly reduced Qmax values. Additionally, other amino acid substitutions were performed at three of the six antigen contact residues (L91Ser, L96Trp and H33Trp) to further evaluate the role of each in Fl binding. Therefore, the following mutations were constructed and characterized: SerL91Asn, TrpL96Tyr, TrpL96Phe, TrpL96Leu, TrpH33Tyr and TrpH33Phe. Results of site-specific mutagenesis studies are discussed in terms of Mab active site structure and suggest that L32Tyr, L91Ser and H33Trp are important for high affinity Fl binding and efficient Fl quenching.
Denzin
L K
LK
Department of Microbiology, University of Illinois at Urbana-Champaign 61801.
Gulliver
G A
GA
Voss
E W
EW
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics*; D001483:Base Sequence; D001666:Binding Sites, Antibody; D001667:Binding, Competitive; D003196:Computer Graphics; D017931:DNA Primers; D019793:Fluorescein; D005452:Fluoresceins / Q000737:chemistry*; D007130:Immunoglobulin Idiotypes / Q000276:immunology; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D017931:DNA Primers; D005452:Fluoresceins; D007130:Immunoglobulin Idiotypes; D019793:Fluorescein