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.
Highlights
Proteins have an indisputable role in key life processes of all organisms
An increased presence of buffer ions on the surface of hen egg-white lysozyme (HEWL) reduces the influence of salt ions (Br−) on effective protein−protein interactions. This result confirms our assumption about the competition of salt and buffer ions for binding to the surface of Different experimentally determined parameters, that are commonly used to evaluate protein−protein interactions in solution, such as second virial coefficient, and interaction diffusion coefficient were shown to depend on the buffer in which the protein solution is prepared, even at the same pH, and same ionic strength of the solution
A closer examination of the buffer properties indicated that the buffer ions bind to the oppositely charged amino-acid residues on the protein surface and in this way reduce the surface charge of the protein molecules that is one of the factors determining the stability of protein formulations
Summary
Proteins have an indisputable role in key life processes of all organisms. They are the most abundant biomolecules in living cells. The highest value of ε0 (indicating strongest attraction between proteins) was obtained for phosphate buffer that has at the same time the lowest Jones-Dole B coefficient (indicating more loosely bound water molecules), which results in the lowest phase stability of HEWL, the lowest value of ε0 was determined for cacodylate, which has the strongest interaction with water, with HEWL demonstrating the highest phase stability under such conditions This trend of binding buffer ions to HEWL surface was further confirmed by measuring the zeta potential of different HEWL−buffer solutions (see Figure S3). This result confirms our assumption about the competition of salt and buffer ions for binding to the surface of HEWL molecules
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