Probing the combining site of an anti-carbohydrate antibody by saturation-mutagenesis: role of the heavy-chain CDR3 residues

Sevenmutants of the heavy chain CDR3 variable loop of the Salmonella B O-polysaccharide-specific antibody Fab fragment were characterized thermodynamically using titration microcalorimetry. Results reveal that the glycine at position 102 cannot be replaced while the glycine at position 100 and the tyrosine at position 103 can be substituted with only small effects on binding. Substitution of the histidine at position 101 with acidic amino acids resulted only in a small reduction in binding affinity. Further, the calorimetric analysis employed revealed tremendous thermodynamic alterations otherwise masked by the similarity in binding constants of the mutants.