Effects of substitutions of closely related amino acids at the contact surface in an antigen-antibody complex on thermodynamic parameters

W Ito,Y Iba,Y Kurosawa

doi:10.1016/s0021-9258(19)85466-2

Abstract

We constructed a library of 512 kinds of Fv fragment, derivatives of a monoclonal antibody, D1.3, specific for hen egg-white lysozyme, in which a total of nine of the original amino acids were replaced by closely related amino acids at positions in the complementarity-determining regions of the H chain. More than 80% of the clones in the library produced Fv fragments in Escherichia coli. Two wild-type and 13 mutant Fv fragments were prepared in large quantities and subjected to analysis by differential titration calorimetry. The association constants of the 15 Fv fragments with hen egg-white lysozyme were distributed between 0.12 x 10(7) and 1.59 x 10(8) M-1. The changes in delta H0 and -T delta S0 caused by one-point mutation at each position did not have intrinsic values for each change. The same changes at one position had different effects on KA, delta H0, and -T delta S0 when differences had been introduced in other regions. The delta(delta G0) caused by a single-point mutation ranged from -0.56 to 1.56 kcal/mol. By contrast, the delta(delta H0) and delta(-T delta S0) caused by a single-point mutation ranged from -3.5 to 3.4 and from -3.8 to 3.4 kcal/mol, respectively. When antibodies gain the binding energy contributed by the effects of enthalpy, they lose the binding energy contributed by the effects of entropy and vice versa. In general, changes in entropy compensate for changes in enthalpy.

Highlights

Effects of Substitutions of Closely Related Amino Acids atthe Contact Surface in an Antigen-Antibody Complex on Thermodynamic Parameters*
The association constants of th1e5 Fv fragments with hen egg-white lysozyme were distributed contact surface is rather large and that more than 15 amino acid residues of each molecule are involved in non-covalent bonds at thecontact surfaces [4,5,6,7,8,9]
The A(AGo) caused Escherichia coli and its complex with the antigen have by a single-pointmutation ranged from-0.56 to 1.56 kcal/mol

Summary

Expression of Fv fragments was examined directly by Western

Primers I and 111 contained an EcoRI site (underlined). The procedure was essentially the same as thatdescribed by and I1 contained a PstI site (underlined). With pWild-TDNA as template brane was soaked in PBS/T thatcontained 1%BSA at room temperand primers I and 1’, 73-bp DNA fragments were amplified. The ature for 1h, washed three timeswith PBS/T, soaked in PBS/T that primers wereremovedby centrifugation in a Centricon 10 system contained D1.3-specific antibody, kept at room temperature for 4 h,. Another PCR with the same template DNA and primers and washed threetimes with PBS/T. After digestion with PstI, bothfragments were ligated, andthe After five washes with PBS/T, bands of polypeptides were detected products were separated by electrophoresis in a 6% polyacrylamide by the ECL Western blotting system (Amersham Corp.)

Molecular form

Light chain

Substitution of amino acids in the mutant antibodies and their characteristics "

Findings

ThermoPdayrnaammeicters of Mutant Antibodies