VanAntwerp J J, Wittrup K D
Department of Chemical Engineering, University of Illinois, Urbana 61801, USA.
J Mol Recognit. 1998 Winter;11(1-6):10-3. doi: 10.1002/(SICI)1099-1352(199812)11:1/6<10::AID-JMR381>3.0.CO;2-H.
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 five-fold 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.
了解抗原-抗体结合中结合自由能的结构和动态决定因素具有重要意义。许多工作集中在选择性突变上，以定位关键相互作用残基，但这通常会导致亲和力降低。本研究转而研究一种高亲和力突变体，以表征亲和力成熟过程的热力学途径。我们比较了抗鸡卵溶菌酶单链抗体scFv D1.3与高亲和力突变体的抗原结合能（霍金斯，R.E.，拉塞尔，S.J.，拜尔，M.和温特，G.（1993年）。《分子生物学杂志》234卷，958 - 964页）。通过等温滴定量热法测量，该突变体对溶菌酶的亲和力高五倍，但结合时的焓和热容变化几乎相同。因此，大部分结合自由能差异可归因于熵效应。用丙烯酰胺进行荧光猝灭表明，这种更有利的熵变化可能源于突变体-溶菌酶复合物更灵活，因此是一种构象熵效应。
