Effect of Buffer on Protein Stability in Aqueous Solutions: A Simple Protein Aggregation Model

Abstract

Liquid-liquid phase separation (LLPS) of proteins has recently been associated with the onset of numerous diseases. Despite several studies in this area of protein aggregation, buffer-specific effects always seem to be overlooked. In this study we investigated the influence of buffers on the phase stability of hen egg-white lysozyme (HEWL) and its respective protein-protein interactions by measuring the cloud point temperature, second virial coefficient, and interaction diffusion coefficient of several HEWL-buffer solutions (MOPS, phosphate, HEPES, cacodylate) at pH 7.0. The results indicate that the buffer molecules, depending on their hydration, adsorb on the protein surface, and modulate their electrostatic stability. The obtained information was used to extend the recently developed coarse-grained protein model to incorporate buffer-specific effects. Treated by Wertheim's perturbation theory the model qualitatively correctly predicted the experimentally observed phase separation of all investigated HEWL-buffer solutions, and further allowed us to predict the phase stability of protein formulations even in experimentally unattainable conditions. Since the theory can be straightforwardly extended to include multiple components it presents a useful tool to study protein aggregation in crowded cell-like systems.

Figure 1

Measured T_cloud values of HEWL at two concentrations in chosen 0.1 M buffers with pH 7.0 extrapolated to zero NaBr concentration, where γ₁ and γ₂ denote 90 and 125 mg/mL HEWL, respectively.

Figure 2

Correlations between measured B₂₂ and k_D with T_cloud values for 90 mg mL^–1 of HEWL in selected 0.1 M buffers with pH 7.0. The straight line was obtained with a best linear least-squares fit to the experimental data.

Figure 3

Coarse-grained model of HEWL with the shown sum of hard-sphere and attractive interactions between proteins.

Figure 4

Comparison of predicted phase diagrams of HEWL in different 0.1 M buffers and pH 7.0 with experimentally obtained T_cloud values extrapolated to zero salt concentration.

Figure 5

Correlation between Jones-Dole coefficients, B, and the square-well depth parameter at zero NaBr concentration, ε₀/k_B, for different 0.1 M buffers at pH 7.0.

Figure 6

Correlation between the average salt-specific parameter a and the average square-well depth parameter at zero NaBr concentration, ε₀.