Salt links dominate affinity of antibody HyHEL-5 for lysozyme through enthalpic contributions

Jamie A. Wibbenmeyer, Peter Schuck, Sandra J. Smith-Gill, Richard C. Willson

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20 Scopus citations


The binding of murine monoclonal antibody HyHEL-5 to lysozyme has been the subject of extensive crystallographic, computational, and experimental investigations. The complex of HyHEL-5 with hen egg lysozyme (HEL) features salt bridges between Fab heavy chain residue Glu50, and Arg45 and Arg68 of HEL. This interaction has been predicted to play a dominant role in the association on the basis of molecular electrostatics calculations. The association of aspartic acid and glutamine mutants at position 50(H) of the cloned HyHEL-5 Fab with HEL and bobwhite quail lysozyme (BQL), an avian variant bearing an Arg68 → Lys substitution in the epitope, was characterized by isothermal titration calorimetry and sedimentation equilibrium. Affinities for HEL were reduced by 400-fold (E50(H)D) and 40,000-fold (E50(H)Q) (ΔΔG°estimated at 4.0 and 6.4 kcal mol-1, respectively). The same mutations reduce affinity for BQL by only 7- and 55- fold, respectively, indicating a reduced specificity for HEL. The loss of affinity upon mutation is in each case primarily due to an unfavorable change in the enthalpy of the interaction; the entropic contribution is virtually unchanged. An enthalpy-entropy compensation exists for each interaction; ΔH°decreases, while ΔS°increases with temperature. The ΔCp for each mutant interaction is less negative than the wild-type. Mutant-cycle analysis suggests the mutations present in the HyHEL-5 Fab mutants are linked to those present in the BQL with coupling energies between 3 and 4 kcal mol-1.

Original languageEnglish (US)
Pages (from-to)26838-26542
Number of pages297
JournalJournal of Biological Chemistry
Issue number38
StatePublished - Sep 17 1999

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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