Thermodynamics of Fab-ssDNA interactions: Contribution of heavy chain complementarity determining region 3

The recombinant anti-ssDNA Fab, DNA-1, and 16 heavy chain complementarity d etermining region 3 (HCDR3) mutant variants were selected for thermodynamic characterization of ssDNA binding. The affinity of Fab to (dT)(15) under d ifferent temperatures and cation concentrations was measured by equilibrium fluorescence quenching titration. Changes in the standard Gibbs free bindi ng energy (Delta G degrees), enthalpy (Delta H degrees), entropy (Delta S d egrees), and the number of ionic pairs (Z) formed upon interaction were det ermined. All Fab possessed an enthalpic nature of interaction with ssDNA, t hat was opposite to the previously reported entropically driven binding to dsDNA [Tanha, J., and Lee, J. S. (1997) Nucleic Acids Res. 25, 1442-1449], The contribution of separate residues of HCDR3 to ssDNA interaction was inv estigated. Analysis of the changes in Delta H degrees and T Delta S degrees , induced by substitutions in HCDR3, revealed a complete entropy/enthalpy c ompensation. Mutations R98A and D108A at the ends of the HCDR3 loop produce d increases in T Delta S degrees by 10.4 and 15.9 kcal/mol, respectively. S ubstitution of proline for arginine at the top of HCDR3 resulted in a new e lectrostatic contact with (dT)15 The observed linear correlation of Z and D elta G degrees of nonelectrostatic interactions (Delta G degrees(nonel)) at the anti-ssDNA combining site was used for the estimation of the specific Delta G degrees(nonel) [-20 to -25 cal/(mol.Angstrom(2))], the average cont act area (450-550 Angstrom(2)), the maximal Z (6-7), and the limit in affin ity under standard cation concentrations [(0.5-1) x 10(8) M-1] for this fam ily of Fab. Results suggested that rational engineering of HCDR3 could be u tilized to control the affinity and likely the specificity of Ab-DNA intera ctions.