Aneta Michna

Theoretical calculations and various experimental techniques were applied to determine fundamental physicochemical characteristics of the RNA-binding protein low complexity domain (hnRNPA2 LCD), in sodium chloride solutions. The protein monomer size, cross-section area, the dependence of the nominal charge on pH, and its isoelectric point were predicted. These theoretical data allowed one to analyze and interpret the adsorption of hnRNPA2 LCD molecules on mica, which was investigated by the streaming potential technique, and on polymer particles, acquired by laser Doppler velocimetry. It was shown that the protein adsorbed in the form of oligomers whose size was resolved by atomic force microscopy. In the case of the adsorption on particles, the oligomer size and zeta potential were derived by applying the general electrokinetic model. Additionally, the electrokinetic properties of the hnRNPA2 LCD functionalized particles were determined and compared with the bulk protein properties. Using these results, a fast and easy method for quantifying the oligomerization kinetic of unstable protein solutions was developed.

Various polycations and polyanions were sequentially adsorbed onto the gold electrode of a quartz crystal microbalance with dissipation monitoring. The study focused on determining the adsorption kinetics, viscoelastic properties, and electroresponsivity of polyelectrolyte layers. For the first time, it was demonstrated that the structure (compact or expanded) of the layers can be determined by electroresponsivity. Viscoelastic modeling alone did not provide a conclusive answer as to whether the layers were compact or expanded. The study was further enriched by streaming potential and contact angle measurements, where polyelectrolyte multilayers were formed on mica. It was found that successive adsorption of layers led to periodic inversion of the zeta potential. Systematic differences were observed between the different top layers, which were explained by intermixing between layers. The presence or absence of interpenetration, as determined by the measurements of streaming potential and contact angles, correlated well with electroresponsivity.

Molecular dynamic modeling and various experimental techniques, including multi-angle dynamic light scattering (MADLS), streaming potential, optical waveguide light spectroscopy (OWLS), quartz crystal microbalance with dissipation (QCM), and atomic force microscopy (AFM), were applied to determine the basic physicochemical parameters of fibroblast growth factor 21 in electrolyte solutions. The protein size and shape, cross-section area, dependence of the nominal charge on pH, and isoelectric point of 5.3 were acquired. These data enabled the interpretation of the adsorption kinetics of FGF 21 on bare and macrocation-covered silica investigated by OWLS and QCM. It was confirmed that the protein molecules irreversibly adsorbed on the latter substrate, forming layers with controlled coverage up to 0.8 mg m⁻², while their adsorption on bare silica was much smaller. The viability of two cell lines, CHO-K1 and L-929, on both bare and macrocation/FGF 21-covered substrates was also determined. It is postulated that the acquired results can serve as useful reference systems for designing complexes that can extend the half-life of FGF 21 in its active state.