Premium
Thermodynamic characterization of affinity maturation: the D1.3 antibody and a higher‐affinity mutant
Author(s) -
VanAntwerp Jennifer J.,
Wittrup K. Dane
Publication year - 1998
Publication title -
journal of molecular recognition
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.401
H-Index - 79
eISSN - 1099-1352
pISSN - 0952-3499
DOI - 10.1002/(sici)1099-1352(199812)11:1/6<10::aid-jmr381>3.0.co;2-h
Subject(s) - isothermal titration calorimetry , lysozyme , mutant , affinity maturation , enthalpy , chemistry , kinetics , förster resonance energy transfer , entropy (arrow of time) , configuration entropy , thermodynamics , biophysics , biochemistry , microbiology and biotechnology , crystallography , fluorescence , biology , gene , physics , quantum mechanics
Understanding the structural and dynamic determinants of binding free energy in the antigen–antibody bond is of great interest. Much work has focused on selective mutations in order to locate key interaction residues, but this generally results in reduced affinity. The present work instead examines a higher‐affinity mutant to characterize the thermodynamic pathway of the affinity maturation process. We have compared the antigen binding energetics of scFv D1.3, an anti‐hen egg lysozyme single chain antibody, with a higher‐affinity mutant (Hawkins, R. E., Russell, S. J., Baier, M. and Winter, G. (1993). J. Mol. Biol. 234 , 958–964). The mutant has fivefold higher affinity for lysozyme but nearly the same enthalpy and heat capacity change upon binding, as measured by isothermal titration calorimetry. Thus, much of the binding free energy difference can be attributed to entropic effects. Fluorescence quenching with acrylamide indicates that this more favorable entropy change may result from a more flexible mutant–lysozyme complex and thus be a configurational entropy effect. Copyright © 1998 John Wiley & Sons, Ltd.