![]() No correlation was observed between decreases in apolar, polar, or aggregate (sum of the apolar and polar) buried surface area in the V L92 mutant series and changes in the enthalpy of formation. Formation of all six mutant complexes is marked by a decrease in binding enthalpy that exceeds the decrease in binding free energy, such that the loss in enthalpy is partly offset by a compensating gain in entropy. The affinities of the mutant Fv fragments for HEL are 10- to 100-fold lower than that of the original antibody. The looses in apolar buried surface area in the mutant complexes, relative to wild type, range from 25 (V LF92) to 115 2 (V LA92), with no significant shifts in the positions of protein atoms at the mutation site for any of the complexes except V LA92, where there is a peptide flip. In the wild-type complex, V LW92 occupies a large hydrophobic pocket on the surface of HEL and constitutes an energetic hot spot for antigen binding. Crystal structures of six FvD1.3-HEL mutant complexes in which an interface tryptophan residue (V LW92) has been replaced by residues with smaller side chains (alanine, serine, valine, aspartate, histidine and phenylalanine) were determined to resolutions between 1.75 and 2.00. We have employed site-directed mutagenesis, X-ray crystallography, and isothermal titration calorimetry to investigate the role of hydrophobic interactions in stabilizing the complex between the Fv fragment of the anti-hen egg white lysozyme (HEL) antibody D1.3 and HEL. Antigen-antibody complexes provide useful models for analyzing the thermodynamics of protein-protein association reactions.
0 Comments
Leave a Reply. |