EFFECTS OF SUBSTITUTIONS OF CLOSELY-RELATED AMINO-ACIDS AT THE CONTACT SURFACE IN AN ANTIGEN-ANTIBODY COMPLEX ON THERMODYNAMIC PARAMETERS

We constructed a library of 512 kinds of Fv fragment, derivatives of a monoclonal antibody, D1.3, specific for hen egg-white lysozyme, in wh ich a total of nine of the original amino acids were replaced by close ly related amino acids at positions in the complementarity-determining regions of the H chain. More than 80% of the clones in the library pr oduced Fv fragments in Escherichia coli. Two wild-type and 13 mutant F v fragments were prepared in large quantities and subjected to analysi s by differential titration calorimetry. The association constants of the 15 Fv fragments with hen egg-white lysozyme were distributed betwe en 0.12 x 10(7) and 1.59 x 10(8) M-1. The changes in DELTAH-0 and -TDE LTAS0 caused by one-point mutation at each position did not have intri nsic values for each change. The same changes at one position had diff erent effects on K(A), DELTAH-0, and -TDELTAS0 when differences had be en introduced in other regions. The DELTA(DELTAG0) caused by a single- point mutation ranged from -0.56 to 1.56 kcal/mol. By contrast, the DE LTA(DELTAH-0) and DELTA(-TDELTAS0) caused by a single-point mutation r anged from -3.5 to 3.4 and from -3.8 to 3.4 kcal/mol, respectively. Wh en antibodies gain the binding energy contributed by the effects of en thalpy, they lose the binding energy contributed by the effects of ent ropy and vice versa. In general, changes in entropy compensate for cha nges in enthalpy.