Single-chain site-specific mutations of fluorescein-amino acid contact residues in high affinity monoclonal antibody 4-4-20.
Previous crystallographic studies of high affinity anti-fluorescein monoclonal antibody 4-4-20 (Ka = 1.7 x 10(10) M-1) complexed with fluorescyl ligand resolved active site contact residues involved in binding. For better definition of the relative roles of three light chain antigen contact residues (L27dhis, L32tyr and L34arg), four site-specific mutations (L27dhis to L27lys, L32tyr to L32phe, and L34arg to L34lys and L34his) were generated and expressed in single-chain antigen binding derivatives of monoclonal antibody 4-4-20 containing two different polypeptide linkers (SCA 4-4-20/205c, 25 amino acids and SCA 4-4-20/212, 14 amino acids). Results showed that L27dhis and L32tyr were necessary for wild type binding affinities, however, were not required for near-wild type Qmax values (where Qmax is the maximum fluoroscein fluorescence quenching expressed as percent). Tyrosine L32 which hydrogen bonds with ligand was also characterized at the haptenic level through the use of 9-hydroxyphenylfluoron which lacks the carboxyl group to which L32 tyrosine forms a hydrogen bond. Results demonstrated that wild type SCA and mutant L32phe possessed similar HPF binding characteristics. Active site contact residue L34arg was important for fluorescein quenching maxima and binding affinity (L34his mutant), however, substitution of lysine for arginine at L34 did not have a significant effect on observed Qmax value. In addition, substitutions had no effect on structural and topological characteristics, since all mutants retained similar idiotypic and metatypic properties. Finally, two linkers were comparatively examined to determine relative contributions to mutant binding properties and stability. No linker effects were observed. Collectively, these results verified the importance of these light chain fluorescein contact residues in the binding pocket of monoclonal antibody 4-4-20.
Denzin
L K
LK
Department of Microbiology, University of Illinois, Urbana 61801.
Whitlow
M
M
Voss
E W
EW
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics; D000915:Antibody Affinity; D001666:Binding Sites, Antibody; D003001:Cloning, Molecular; D003196:Computer Graphics; D004252:DNA Mutational Analysis; D019793:Fluorescein; D005452:Fluoresceins / Q000737:chemistry; D007130:Immunoglobulin Idiotypes / Q000737:chemistry; D066298:In Vitro Techniques; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D005452:Fluoresceins; D007130:Immunoglobulin Idiotypes; D019793:Fluorescein