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Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope
Published on: February 28, 2019
Kinetic analysis of the interaction between protein A domain variants and human Fc using plasmon resonance detection
L Jendeberg1, B Persson, R Andersson
1Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
Insights
Biospecific interaction analysis revealed that a divalent staphylococcal protein A analogue (ZZ) binds human immunoglobulin G1 (Fc1) with higher affinity. Mutating the Z analogue altered binding kinetics, with some variants showing significantly reduced association rates.
Area of Science:
- Biochemistry
- Molecular Biology
- Protein Engineering
Background:
- Staphylococcal protein A analogues are crucial for studying protein interactions.
- Understanding the binding kinetics of Fc portion of human immunoglobulin G1 (Fc1) is vital for therapeutic antibody development.
- The Fc1-binding domain (Z) of staphylococcal protein A and its variants offer a model system for analyzing protein-ligand interactions.
Purpose of the Study:
- To investigate the specific binding interactions between Fc1 and monovalent (Z) and divalent (ZZ) staphylococcal protein A analogues.
- To characterize the binding kinetics and affinity of five single amino acid substituted Z variants.
- To compare binding data obtained from biospecific interaction analysis (BIA) with a competitive displacement assay.
Main Methods:
- Real-time biospecific interaction analysis (BIA) was employed to measure binding parameters.
- Association rate constants (kon), dissociation rate constants (koff), and affinity constants (Kaff) were calculated.
- Competitive displacement assays were used for comparison with BIA data.
Main Results:
- The divalent ZZ analogue exhibited higher affinity for Fc1, primarily due to a slower dissociation rate (koff).
- Four out of five Z variants showed reduced Fc1 affinity; two (L17D, I31A) had decreased association rates (kon), while two others (N28A, K35A) showed increased dissociation rates (koff).
- BIA and competitive displacement assay results generally agreed for most variants, except for Z(I31A), where a significant discrepancy in kon was observed.
Conclusions:
- Divalent ZZ analogue enhances binding affinity to Fc1 through kinetic stabilization.
- Single amino acid substitutions in the Z analogue can significantly impact binding kinetics and affinity.
- Discrepancies between BIA and competitive displacement assays highlight the importance of method selection for characterizing protein interactions, particularly for variants with drastically altered kinetics.
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 (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.
