The anti-fluorescein antibody active site: A single-chain site-specific mutagenesis study
Denzin, Lisa Kae
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Permalink
https://hdl.handle.net/2142/21067
Description
Title
The anti-fluorescein antibody active site: A single-chain site-specific mutagenesis study
Author(s)
Denzin, Lisa Kae
Issue Date
1992
Doctoral Committee Chair(s)
Voss, Edward W., Jr.
Department of Study
Microbiology
Discipline
Microbiology
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Microbiology
Health Sciences, Immunology
Language
eng
Abstract
Crystallographic studies of high affinity anti-fluorescein monoclonal antibody 4-4-20 (K$\sb{\rm a}$ = 1.7 10$\sp{10}$ M$\sp{-1}$) complexed with fluorescyl ligand defined the six antibody active site contact residues involved in FI binding. For better definition of the relative role of each antigen contact residue in high affinity FI binding, each contact residue was changed to various amino acids in the single chain derivative of Mab 4-4-20 and following expression in Escherichia coli, denaturation, refolding and purification, each SCA (single chain antibody) mutant was characterized in terms of FI binding affinity, Q$\sb{\rm max}$ (maximum fluorescein fluorescence quenching), $\lambda\sb{\rm max}$ (Absorption maxima) and idiotype. Alanine substitutions at the six ligand-contact residues reduced the SCA binding affinities and quenching maxima for all the Ala mutants except L27d which retained wild type binding characteristics. Results of Ala substitution mutagenesis suggested that L32$\sp{\rm Tyr}$, L91$\sp{\rm Ser}$ and H33$\sp{\rm Trp}$ are most important for high affinity FI binding and efficient FI quenching. Substitution of Tyr and Phe at residue H33 resulted in binding affinities that were greater than that obtained from the H33$\sp{\rm Ala}$ mutant and suggested that another aromatic amino acid could substitute for Trp this residue. A phenylalanine substitution at L32$\sp{\rm Tyr}$, which disrupted the Tyr hydrogen bond with fluorescyl ligand, resulted in a decreased the binding affinity ($\sim$30-fold), but did not effect the quenching maxima. Finally, other amino acid substitutions at L34$\sp{\rm Arg}$, L91$\sp{\rm Ser}$ and L27d$\sp{\rm His}$ resulted in SCA mutants that possessed lower binding affinities and quenching maxima than that obtained for the respective Ala mutant.
Primary structure comparisons of idiotypically cross-reactive Mabs 4-4-20, 9-40, 12-40 and 5-14 possessed identical FI contact residues with the exception of L34His for L34Arg. Site specific mutagenesis studies of SCA 4-4-20 in which L34Arg was changed to L34His resulted in $\sim$1000-fold and 3-fold decrease in binding affinity and Q$\sb{\rm max}$, respectively, which suggested that L34Arg was responsible for 4-4-20 increased binding affinity and fluorescence quenching. Collectively, these results suggest that the combining sites of Mab 9-40, 12-40 and 5-14 may possess different active site structures than Mab 4-4-20. (Abstract shortened by UMI.)
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