Thermodynamics of Protein-Protein Interaction Studied by Using BIAcore and Single-Site Mutagenesis

Abstract

Protein engineering strategies for mapping the functional epitopes of interacting molecules require rapid and accurate methods for measuring binding constants. Results with two systems, a humanized antibody (hu4D5) binding to antigen and coagulation factor VII binding to the cofactor protein tissue factor, are used to describe the use of biosensor technology in mapping protein-protein interfaces. Affinity measurements on the BlAcore are fast and highly reproducible. Binding constants measured by this technique agree with values determined by other methods. For the hu4D5 antibody, results of BlAcore and calorimetric measurements on single alanine mutants show that a few residues in the binding interface dominate the energetics of antigen binding. These mutations primarily affect the off-rate for binding. A complete thermodynamic analysis of antigen binding to these mutant proteins highlights the importance of the hydrophobic effect for binding. These results clearly show that the BlAcore instrument is a powerful tool for evaluating protein-protein interactions.