Objective

Protein interactions with living cells are complex and often heterogeneous due to dimerization of receptors, modifications, avidity effects and ligands with several built-in properties. Information of kinetic properties are needed to resolve such complex interactions. It is in the curvature of the binding we find that information. We have used LigandTracer® for analysis of EGF interactions with living cells to generate binding curves of protein interactions. We have also analyzed interactions of a Fab fragment towards CD44v6 with different cell lines and after different label modifications. Evaluation of the information can be performed by classical biophysical models but often the curve fit is not good enough due to the complexity of the interaction.  For such interactions we use Interaction Map® to identify the most important parallel interaction processes that build the complex binding curve. Each process is presented as a peak in the general on-off plot. It gives us on-rate and off-rate kinetic constants of the processes and how much each contribute to the binding curve in a parameter Weight. SPR data from protein-protein interactions can also be analyzed by Interaction Map. Avidity effects due to solid phase assays can be separated from the affinity.  More complex analysis of a bifunctional Affibody® molecules have more interaction processes than expected.