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

Abstract

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 (B₂₃) 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 B₂₃ between lysozyme (Lys) and bovine serum albumin (BSA) and between Lys and protein isolates (whey and canola). We find that B₂₃ 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.

Figure 1

Characteristic elution profiles measured by the CIC approach monitoring the interactions of Lys-BSA (purple) in comparison to the self-interacting Lys-Lys (black) (A) and of Lys-WPI BLG (blue), Lys-WPI ALAC (green), and Lys-CPI NAP (orange) in comparison to the self-interacting Lys-Lys (dash-dotted black line) (B) in 50 mM sodium phosphate buffer at pH ∼ 6.9.

Figure 2

Change of B₂₃ (ΔB₂₃) for the interaction between lysozyme grafted to the column and BSA (at 20 mg/mL) in the presence of added proline dissolved in 50 mM sodium phosphate buffer at pH ∼ 6.9. The error bars reflect the measurement uncertainties (std of ΔB₂₃).

Figure 3

ΔB₂₃ for the interaction between lysozyme, grafted to the column, and the two whey protein isolates, WPI BLG (circles) and WPI ALAC (squares), and the canola protein isolate, CPI NAP (triangles), is displayed for different amino acids (arginine (blue), glutamine (green), and proline (red)) at 1–100 mM dissolved in the protein injected and in the 50 mM sodium phosphate buffer at pH ∼ 6.9. The error bars reflect the measurement uncertainties (std of ΔB₂₃).

Figure 4

Normalized CD curves for the individual proteins of lysozyme (A) and WPI BLG (B) at ∼25 μM and for the interacting Lys-WPI BLG sample (C) at ∼50 μM in buffer and in the presence of 1 mM proline and 10 mM proline and 10 mM glutamine. In (D), the calculated wavelength (λ) ratio between the CD signal measured at λ = 222 nm versus λ = 208 nm for the Lys and WPI BLG measured separately and together in buffer solutions (gray) and in the presence of 1 mM proline (light green) and 10 mM proline (dark green) and 10 mM glutamine (orange).

Figure 5

(A) Representative steady-state affinity fitting for the SPR response of WPI BLG interacting with lysozyme in PBS yielding a K_D value of (1.4 ± 0.2) mM which is indicated by the dashed line. (B) Steady-state affinity fitting for a representative SPR response for WPI BLG interacting with lysozyme in the presence of 10 mM arginine yielding a K_D of (5 ± 1) M as indicated by the dashed line.