Modulating Weak Protein-Protein Cross-Interactions by the Addition of Free Amino Acids at Millimolar Concentrations.

In this paper, we quantify weak protein-protein interactions in solution using cross-interaction chromatography (CIC) and surface plasmon resonance (SPR) and demonstrate that they can be modulated by the addition of millimolar concentrations of free amino acids. With CIC, we determined the second osmotic virial cross-interaction coefficient () as a proxy for the interaction strength between two different proteins. We perform SPR experiments to establish the binding affinity between the same proteins. With CIC, we show that the amino acids proline, glutamine, and arginine render the protein cross-interactions more repulsive or equivalently less attractive. Specifically, we measured between lysozyme (Lys) and bovine serum albumin (BSA) and between Lys and protein isolates (whey and canola). We find that increases when amino acids are added to the solution even at millimolar concentrations, corresponding to protein/ligand stoichiometric ratios as low as 1:1. With SPR, we show that the binding affinity between proteins can change by 1 order of magnitude when 10 mM glutamine is added. In the case of Lys and one whey protein isolate (WPI), it changes from the mM to the M range, thus by 3 orders of magnitude. Interestingly, this efficient modulation of the protein cross-interactions does not alter the protein's secondary structure. The capacity of amino acids to modulate protein cross-interactions at mM concentrations is remarkable and may have an impact across fields in particular for specific applications in the food or pharmaceutical industries.