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 determining region 3 (HCDR3) mutant variants were selected for thermodynamic characterization of ssDNA binding. The affinity of Fab to (dT)(15) under different temperatures and cation concentrations was measured by equilibrium fluorescence quenching titration. Changes in the standard Gibbs free binding energy (DeltaG degrees ), enthalpy (DeltaH degrees ), entropy (DeltaS degrees ), and the number of ionic pairs (Z) formed upon interaction were determined. All Fab possessed an enthalpic nature of interaction with ssDNA, that 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 investigated. Analysis of the changes in DeltaH degrees and TDeltaS degrees, induced by substitutions in HCDR3, revealed a complete entropy/enthalpy compensation. Mutations R98A and D108A at the ends of the HCDR3 loop produced increases in TDeltaS degrees ( )()by 10.4 and 15.9 kcal/mol, respectively. Substitution of proline for arginine at the top of HCDR3 resulted in a new electrostatic contact with (dT)(15). The observed linear correlation of Z and DeltaG degrees ( )()of nonelectrostatic interactions (DeltaG degrees (nonel)) at the anti-ssDNA combining site was used for the estimation of the specific DeltaG degrees (nonel) [-20 to -25 cal/(mol.A(2))], the average contact area (450-550 A(2)), the maximal Z (6-7), and the limit in affinity under standard cation concentrations [(0.5-1) x 10(8) M(-)(1)] for this family of Fab. Results suggested that rational engineering of HCDR3 could be utilized to control the affinity and likely the specificity of Ab-DNA interactions.
Komissarov
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Department of Biochemistry, University of Missouri School of Medicine, Columbia, Missouri 65212, USA.
Deutscher
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SL
D000818:Animals; D000974:Antibodies, Antinuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D007140:Immunoglobulin Fab Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity; D013816:Thermodynamics
D000974:Antibodies, Antinuclear; D004277:DNA, Single-Stranded; D007140:Immunoglobulin Fab Fragments; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins