High resolution functional analysis of antibody-antigen interactions

Jin, Lei; Fendly, Brian M.; Wells, James A.

doi:10.1016/0022-2836(92)90636-x

Cited by 213 publications

(137 citation statements)

References 39 publications

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“…Since its introduction by Wells (1991), alanine scan mutagenesis is finding increasing application in the investigation of proteinprotein complexes (Jin et al, 1992;Cunningham & Wells, 1993;Dall'Acqua et al, 1996;Pearce et al, 1997). While elegant in its simplicity, a single substitution may yield results that give an incomplete measure of how a given residue contributes to the stability of a complex.…”

Section: Limitations Of Alanine Scan Mutagenesismentioning

confidence: 99%

“…Immunochemical applications include inter alia human growth hormone-antibody interactions (Jin et al, 1992) and antiidiotypic antibody E5.2 and HEWL-antibody Dl .3 interactions (Dall'Acqua et al, 1996). We show here that alanine scan mutagenesis needs to be supplemented with additional mutations in order to understand fully the role of a given residue.…”

mentioning

confidence: 99%

“…The achievement of a quantitative comprehension of the contributions of the individual amino acid side chains to the stability and kinetics associated with complex formation is critical to a full understanding of these phenomena. Recently, a number of such directed investigations have been focused on antigen-antibody complexes Reprint requests to: Jack F, Kirsch (Jin et al, 1992;Kam-Morgan et al, 1993;Kelley & O'Connell, 1993;Dall'Acqua et al, 1996;Smith-Gill, 1996;Goldman et al, 1997).…”

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confidence: 99%

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Quantitative evaluation of the chicken lysozyme epitope in the HyHEL‐10 fab complex: Free energies and kinetics

1998

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The hen (chicken) egg-white lysozyme (HEWL) epitope for the monoclonal antibody HyHEL-IO Fab (Fab-IO) was investigated by alanine scan mutagenesis. The association rate constants (/con) for the HEWL.Fab-10 complexes were obtained from the homogenous solution method described in the preceding paper (Taylor et al., 1998). A new method for determining the dissociation rate constant (kOff) for the complex, by trapping nascent free antibody with an inactive HEWL mutant is described. The values of k,, fall within a factor of 2 of the wild-type (WT) HEWL value (1.43 f 0.13 X lo6 M" s-'), while the increases in kc,w more nearly reflect the total change in free energies of the complex (AAG"). The dissociation constants (KO) were measured directly in those cases where satisfactory kinetic data could not be obtained. The Y20A, K96A, and K97A HEWL.Fab-IO complexes are destabilized by more than 4 kcal/mol compared to the WT complex. The R21A, L75A, and DlOlA antibody complexes are moderately destabilized (0.7 < AAGo 5 1 .O kcal/mol). Additional mutations of the "hotspot" residues (TYRO, Lys96, Lys97) were constructed to probe, more precisely, the nature of their contributions to complex formation. The results show that the entire hydrocarbon side chains of Tyr20 and Lys97, and only the s-amino group of Lys96, contribute to the stability of the complex. The value of AAG,] for the R21A mutant complex is a distinct outlier in the Arg2l replacement series demonstrating the importance of supplementing alanine scan mutagenesis with additional mutations.

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Section: Limitations Of Alanine Scan Mutagenesismentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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Quantitative evaluation of the chicken lysozyme epitope in the HyHEL‐10 fab complex: Free energies and kinetics

1998

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“…Mutation to Ala of residues other than Gly or Pro eliminates the targeted side chain beyond C␤ and disrupts any interaction that involves that side chain, without introducing new interactions and with the lowest probability of altering the conformation of the polypeptide backbone (5,6). A few groups have applied this approach for a systematic thermodynamic dissection of interfaces in dimeric heterocomplexes (7)(8)(9)(10)(11)(12)(13)(14)(15)(16) or small homo-oligomers (17)(18)(19)(20)(21). These studies have substantially advanced the molecular understanding of protein-protein recognition.…”

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confidence: 99%

Thermodynamic Dissection of a Low Affinity Protein-Protein Interface Involved in Human Immunodeficiency Virus Assembly

Álamo¹,

Neira

Mateu³

2003

Journal of Biological Chemistry

102

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“…Examination of structurally well refined antibody-antigen interactions (11,12) by site-specific amino acid substitutions of antigen and antibody is essential to the further discussion of antibody specificity. Recent advances in phage-display technology will enable us to engineer an antibody molecule for the purposes of grafting the desired specificity and affinity (13)(14)(15).…”

mentioning

confidence: 99%

Thermodynamic Consequences of Grafting Enhanced Affinity toward the Mutated Antigen onto an Antibody

Nishimiya¹,

Tsumoto²,

Shiroishi³

et al. 2000

Journal of Biological Chemistry

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In order to address the mechanism of enhancement of the affinity of an antibody toward an antigen from a thermodynamic viewpoint, anti-hen lysozyme (HEL) antibody HyHEL-10, which also recognize the mutated antigen turkey lysozyme (TEL) with reduced affinity, was examined. Grafting high affinity toward TEL onto Hy-HEL-10 was performed by saturation mutagenesis into four residues (Tyr 53 , Ser 54 , Ser 56, and Tyr 58 ) in complementarity-determining region 2 of the heavy chain (CDR-H2) followed by selection with affinity for TEL. Several clones enriched have a Phe residue at site 58. Thermodynamic analyses showed that the clones selected had experienced a greater than 3-fold affinity increase toward TEL in comparison with wild-type Fv, originating from an increase in negative enthalpy change. Substitution of HyHEL-10 HTyr 58 with Phe led to the increase in negative enthalpy change and to almost identical affinity for TEL in comparison with mutants selected, indicating that mutations at other sites decrease the entropy loss despite little contribution to the affinity for TEL. These results suggest that the affinity of an antibody toward the antigen is enhanced by the increase in enthalpy change by some limited mutation, and excess entropy loss due to the mutation is decreased by other energetically neutral mutations.

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High resolution functional analysis of antibody-antigen interactions

Cited by 213 publications

References 39 publications

Quantitative evaluation of the chicken lysozyme epitope in the HyHEL‐10 fab complex: Free energies and kinetics

Quantitative evaluation of the chicken lysozyme epitope in the HyHEL‐10 fab complex: Free energies and kinetics

Thermodynamic Dissection of a Low Affinity Protein-Protein Interface Involved in Human Immunodeficiency Virus Assembly

Thermodynamic Consequences of Grafting Enhanced Affinity toward the Mutated Antigen onto an Antibody

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