Single-chain site-specific mutations of fluorescein-amino acid contact residues in high affinity monoclonal antibody 4-4-20

Denzin, Lisa K.; Whitlow, M; Voss, Edward W.

doi:10.1016/s0021-9258(18)92814-0

Cited by 51 publications

(16 citation statements)

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“…This decrease was attributed to the ionization of Tyr-L32, which probably disrupts a hydrogen bond between Tyr's hydroxyl group and FI's phenylcarboxylate group. Both of these observations were consistent with AAG°values determined by site-specific mutagenesis studies of the 4-4-20 single-chain antibody (Denzin et al, 1991;Denzin & Voss, 1992). Taken together, these electrostatic interactions account for -4.5 kcal/mol.…”

Section: Discussionsupporting

confidence: 85%

“…The crystal structure of 4-4-20 showed that one of FI's enolic groups (O3) is sufficiently close (2.8 A) to Arg-L34 to form a salt link (Herron et al, 1989(Herron et al, , 1993. Furthermore, site-specific mutagenesis experiments with single-chain derivatives of 4-4-20 have demonstrated that Arg-L34 (when changed to His) was responsible for a decrease in affinity (Denzin et al, 1991). This result indicated that the electrostatic interaction between Arg-L34 and FI's enolic group is an important component of 4-4-20's high antigen binding affinity.…”

Section: Resultsmentioning

confidence: 97%

“…We also studied the conformation of the active site after the ionization of the phenolic group of Tyr-L32 using time-resolved fluorescence spectroscopy. These results are compared to those obtained by chain reassociation studies and site-specific mutagenesis studies (Bedzyk & Voss, 1991;Bedzyk et al, 1992;Denzin et al, 1991).…”

mentioning

confidence: 95%

“…Also, antihapten antibodies are typically used because it is much easier to measure the antigen-binding affinity for a hapten than for a protein antigen. One such study examined the energetics of key contact residues in a series of recombinant single-chain antibodies (SCA) generated from a monoclonal antibody (4-4-20) which binds FI (Denzin et al, 1991;Denzin & Voss, 1992). Since the active site of 4-4-20 and other genetically related anti-FI antibodies (9-40, 12-40, and 5-14) contain hydrophobic, polar, and charged groups (Bedzykef al., 1990), these antibodies comprise an excellent model system for studying the relative contributions of the hydrophobic and electrostatic effects.…”

mentioning

confidence: 99%

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

Omelyanenko

Jiskoot²,

Herron³

1993

Biochemistry

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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)

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Section: Discussionsupporting

confidence: 85%

Section: Resultsmentioning

confidence: 97%

mentioning

confidence: 95%

mentioning

confidence: 99%

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

Omelyanenko

Jiskoot²,

Herron³

1993

Biochemistry

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“…Single-Chain Antibody 4-4-20. SCA was constructed and expressed as described elsewhere (Bird et al, 1988;Denzin et al, 1991). Isolation of SCA was obtained by extracting inclusion bodies accumulated in Escherichia coli cells.…”

Section: Methodsmentioning

confidence: 99%

Pressure-induced dissociation of fluorescein from the anti-fluorescein single-chain antibody 4-4-20

Coelho‐Sampaio

Voss²

1993

Biochemistry

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Hydrostatic pressure was used to dissociate fluorescein (Fl) from the high-affinity anti-Fl single-chain antibody 4-4-20 (SCA 4-4-20). Fl dissociation was monitored by measuring (1) the shift in the Fl absorption peak, (2) the recovery in Fl fluorescence intensity, which is quenched upon SCA binding, or (3) the decrease in Fl fluorescence polarization. Pressure effects were studied at two different Fl:SCA 4-4-20 molar ratios: 1:1, at which Fl fluorescence quenching was ca. 35% at atmospheric pressure, and 1:5, at which quenching reached 95-97% under the same conditions. In both cases, pressure-induced dissociation was favored by concomitant dilution of protein and ligand. Dissociation constants (KD) at each pressure were calculated on the basis of measurements of Fl fluorescence polarization under pressure. The dependence of KD, and consequently of delta G of dissociation, on pressure permitted calculation of the magnitude of the standard volume change (delta V) involved in the dissociation process. According to this study, delta V of dissociation for the Fl-SCA complex is -50 mL/mol, which corresponds to a 10-times higher value than that found for dissociation of Fl from the intact IgG mAb 4-4-20 [Herron, J. N., Kranz, D. M., Jameson, D. M., & Voss, E. W., Jr. (1986) Biochemistry 25, 4602-4609]. This difference is explained in terms of a higher overall flexibility of unliganded SCA and of a less stable binding site in SCA relative to mAb.(ABSTRACT TRUNCATED AT 250 WORDS)

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The structural repertoire of the human V kappa domain.

et al. 1995

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In humans, the gene for the V kappa domain is produced by the recombination of one of 40 functional V kappa segments and one of five functional J kappa segments. We have analysed the sequences of these germline segments and of 736 rearranged V kappa genes to determine the repertoire of main chain conformations, or canonical structures, they encode. Over 96% of the sequences correspond to one of four canonical structures for the first antigen binding loop (L1) and one canonical structure for the second antigen binding loop (L2). Junctional diversity produces some variation in the length of the third antigen binding loop (L3) and in the identity of residues at the V kappa‐J kappa join. However, this is limited and 70% of the rearranged sequences correspond to one of three known canonical structures for the L3 region. Furthermore, we show that the canonical structures selected during the primary response are conserved during affinity maturation: the key residues that determine the conformations of the antigen binding loops are unmutated or undergo conservative mutation. The implications of these results for immune recognition are discussed.

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Single-chain site-specific mutations of fluorescein-amino acid contact residues in high affinity monoclonal antibody 4-4-20

Cited by 51 publications

References 1 publication

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

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

Pressure-induced dissociation of fluorescein from the anti-fluorescein single-chain antibody 4-4-20

The structural repertoire of the human V kappa domain.

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