Kinetic analysis of the interaction between protein a domain variants and human Fc using plasmon resonance detection

Abstract

A real time biospecific interaction analysis (BIA) was performed to study the specific interaction between the Fc portion of human immunoglobulin G₁ (Fc₁) and a one domain analogue (designated Z) of staphylococcal protein A, in monovalent (Z) and divalent (ZZ) forms, and five different single amino acid substituted Z variants (L17D, N28A, F30A, I31A, K35A). Experimental BIA data were used to calculate association rate constants (K_on), dissociation rate constants (k_off). The divalent form (ZZ) showed a higher affinity for Fc₁ mainly because of a slower off rate. Out of the five mutant Z protein, four (L17D, N28A, I31A, K35A) four had the major effect to Fc₁ compared to the parent Z molecule. Surprisingly, two (L17D, I31A) of these four had the major effect of a decreased binding energy as a lowered k_on while the other two (N28A, K35A) mutant proteins showed an increased k_off as the major kinetic difference from Z in their binding to Fc₁. For five of the six different Z variants, as well as for the ZZ molecule, calculated k_aff and calculated differences in binding free energies relative to the parent Z molecule (ΔΔG), are in good agreement with the corresponding values obtained in a competitive displacement assay using radioactively labeled Z as a tracer (Cedergren et al., (1993) Prot. Eng. 6, 441-448). However, the I31A variant, with a measured k_on that was more than three orders of magnitude lower than that of Z in the BIA assay, showed a significant weaker affinity to Fc_I when calculated from BIA data compared ot the competitive displacement assay. The discrepancy between these two methods for Z(I31A) is discussed as well as possible explanations for the unexpected large effect of lowered k_on for two of the mutant Z proteins.