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 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 (kon), dissociation rate constants (koff) and affinity constants (Kaff). The divalent form (ZZ) showed a higher affinity for Fc1 mainly because of a slower off rate. Out of the five mutant Z proteins, four (L17D, N28A, I31A, K35A) showed a decreased affinity 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 kon while the other two (N28A, K35A) mutant proteins showed an increased koff 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 Kaff 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 kon that was more than three orders of magnitude lower than that of Z in the BIA assay, showed a significantly weaker affinity to Fc1 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 kon for two of the mutant Z proteins.
Jendeberg
L
L
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
Persson
B
B
Andersson
R
R
Karlsson
R
R
Uhlén
M
M
Nilsson
B
B
D000595:Amino Acid Sequence; D015374:Biosensing Techniques; D015704:CD4 Antigens / Q000276:immunology / Q000378:metabolism; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding / Q000276:immunology; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015203:Reproducibility of Results; D013205:Staphylococcal Protein A / Q000235:genetics / Q000276:immunology / Q000378:metabolism*
D015704:CD4 Antigens; D007141:Immunoglobulin Fc Fragments; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A