KINETIC-ANALYSIS OF THE INTERACTION BETWEEN PROTEIN-A DOMAIN VARIANTS AND HUMAN FC USING PLASMON RESONANCE DETECTION

A real-time biospecific interaction analysis (BIA) was performed to study the specific interaction between the Fc portion of human immunoglobulin G(1) (Fc(1)) 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)) and affinity constants (K-aff). The divalent form (ZZ) showed a higher affinity for Fc(1) mainly because of a slower off rate. Out of the five mutant Z proteins, four (L17D, N28A, I31A, K35A) showed a decreased affinity to Fc(1) 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 Fc(1). 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 (Delta Delta G), 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 tower than that of Z in the BIA assay, showed a significantly weaker affinity to Fc(1) when calculated from the BIA data compared to 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.