Thermodynamic consequences of grafting enhanced affinity toward the mutated antigen onto an antibody. The case of anti-lysozyme antibody HyHEL-10

Yoshiyuki Nishimiya, Kouhei Tsumoto, Mitsunori Shiroishi, Katsuhide Yutani, Izumi Kumagai

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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 HyHEL-10 was performed by saturation mutagenesis into four residues (Tyr53, Ser54, Ser56, and Tyr58) 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 HTyr58 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.

Original languageEnglish
Pages (from-to)12813-12820
Number of pages8
JournalJournal of Biological Chemistry
Issue number17
Publication statusPublished - Apr 28 2000


All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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