Matej Jaklin

Amyloids are protein aggregates involved in various protein condensation diseases. Our study aims to investigate the influence of glycine on the fibrillization mechanism of β-lactoglobulin (BLG), a model protein known to form amyloid fibrils from hydrolysed peptides in low pH aqueous solutions. We conducted atomistic molecular dynamics simulations of aqueous solutions of native and unfolded BLG in glycine buffer at pH 2.0. During the simulations we put our focus on analysing protein-protein/buffer interactions, structural electrostatic potential mapping, and the residence times of glycine and glycinium near specific amino acid residues. Glycinium cations were found to preferentially interact with specific protein residues potentially masking the outer disulfide bonds, affecting thiol deprotonation and influencing disulfide scrambling equilibrium. These interactions can potentially hinder hydrolysis and change the fibrillization pathway. Further investigations, such as constant pH MD simulations, simulations on disulfide bounded oligomers are warranted to validate these findings and deepen our understanding of protein aggregation mechanisms.

Pharmaceutical design of protein formulations aims at maximum efficiency (protein concentration) and minimum viscosity. Therefore, it is important to know the nature of protein-protein interactions and their influence on viscosity. In this work, we investigated the dependence of the viscosity of BSA in an aqueous 20 mM acetate buffer at pH = 4.3 on protein concentration and on temperature (5–45 °C). The viscosity of the solution increased with protein concentration and was 230% higher than the viscosity of the protein-free formulation at 160 mg/mL. The viscosity decreased by almost 60% in the temperature range from 5 to 45 °C. The agreement of the modified Arrhenius theory with experiment was quantitative, whereas a hard-sphere model provided only a qualitative description of the experimental results. We also investigated the viscosity of a 100 mg/mL BSA solution as a function of the concentration of added low molecular weight salts (LiCl, NaCl, KCl, RbCl, CsCl, NaBr, NaI) in the range of salt concentrations up to 1.75 mol/L. In addition, the particle size and zeta potential of BSA-salt mixtures were determined for solutions containing 0.5 mol/L salt. The trends with respect to the different anions followed a direct Hofmeister series (Cl⁻ > Br⁻ > I⁻), whereas for cations in the case of viscosity the indirect Hofmeister series was observed (Li⁺ > Na⁺ > K⁺ > Rb⁺ > Cs⁺), but the values of particle sizes and zeta potential did not show cation-specific effects. Since the protein is positively charged at pH = 4.3, anions are more attracted to the protein surface and shield its charge, while the interaction with cations is less pronounced. We hypothesize that salt surface charge shielding reduces protein colloidal stability and promotes protein aggregate formation.