Role of electrostatic interactions in the binding of fluorescein by anti-fluorescein antibody 4-4-20

Abstract

Anti-fluorescein antibodies are excellent model systems for studying the biochemical basis of molecular recognition because a prodigious amount of both physico-chemical and structural information is available for these antibodies. Furthermore, recombinant single-chain antibodies have been produced for several anti-fluorescein antibodies, and site-specific mutagenesis studies have defined the energetic contributions of a number of key active-site residues. In previous studies, we determined the three-dimensional structure of an antigen-binding fragment of a high-affinity anti-fluorescein antibody (4-4-20) in complex with fluorescein. These studies showed that fluorescein binds tightly in an aromatic slot and participates in a network of electrostatic interactions. In this report, we examine the role of electrostatic interactions in the 4-4-20 antigen-combining site by observing the effects of pH on the fluorescence of fluorescein and antigen-binding affinity. These studies showed that the salt link between fluorescein and Arg-L34 in 4-4-20 probably accounts for about -1.5 kcal/mol-1 of the observed free energy of interaction. Furthermore, at pH 10 and higher, the affinity decreases by more than 100-fold (delta delta G degrees approximately equal to 3 kcal mol-1). We attributed this decrease to the ionization of Tyr-L32, which probably disrupts a hydrogen bond between tyrosine's hydroxyl group and fluorescein's phenylcarboxylate group. The fluorescence lifetime of the 4-4-20/fluorescein complex was determined at both pH 8 and pH 10.6. Only one lifetime component (0.38 ns) was observed at pH 8, while two components (0.3 and 3.4 ns) were observed at pH 10.6.(ABSTRACT TRUNCATED AT 250 WORDS)

Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity

“…1 A). Electrostatic steering of fluorescein into the binding pocket (23,24) is evidenced by the salt dependence of k ass . The association rate constant of 4 M5.3 is Ϸ14-fold lower than that of 4-4-20 scFv in both PBS and LSB.…”

Molecular evolution of affinity and flexibility in the immune system

“…The Ab 4-4-20 is particularly useful in this regard, as it binds to the chromophore fluorescein (FLU) with high affinity (8). Interaction of 4-4-20 with FLU has greatly facilitated structural, kinetic and, thermodynamic characterization of this Ab (9)(10)(11)(12). Romesberg and colleagues (13)(14)(15) have characterized evolution of the immunological response for the well studied 4-4-20.…”

Application of Fluorescence Spectroscopy for Determining the Structure and Function of Proteins

“…This structure revealed that there were two tryptophan residues in intimate contact with the fluorescein moiety, either of which could participate in formation of a charge transfer complex. Nevertheless, a recent study by Omelyanenko et al (1993) showed that other factors are also involved in the quenching of fluorescein, including the local pH of the antigen-combining site and the formation of a salt link with a buried arginine residue. Several other physicochemical parameters of the antifluorescein system have been investigated using fluorescence quenching.…”