Thermodynamic Investigation of Charge-Charge Interactions in Biological Systems: Protein-Polyelectrolyte Complex Formation and Protein Unfolding

Abstract

The present study investigates the protein-polyelectrolyte complex formation and folding of proteins with a primary focus on how external factors such as temperature, salt concentrations, types of salt ions, reducing agents and pH affect the thermodynamic parameters. Using an array of biophysical techniques, including ITC, nanoDSF, DLS, DSC, CD and pressure-dependent FT-IR, this study provides a comprehensive thermodynamic perspective on protein stability and protein interactions with polyelectrolytes. To further quantify these protein-polyelectrolyte interactions and characterize protein behaviour under varying conditions, theoretical models based on the experimental data were validated and applied. The first part of this thesis contributes to the understanding of how ion-specific effects influence the thermodynamics of protein-polyelectrolyte interactions. The interaction of heparin and lysozyme in the presence of KGlu under varying salt concentrations was measured by ITC. The free energy of binding ΔGb depends strongly on salt concentration cs, with counterion release identified as the primary driving force for the interaction. In this investigation, a model for the free energy of binding ΔGb as the function of the two decisive variables temperature T and salt concentration cs was applied. This model highlights the significant role of hydration in the binding process. It was shown that temperature T, salt concentration cs and the type of ions present in the solution influence the free energy of binding ΔGb. In the second part, a critical comparison of Differential Scanning Fluorimetry nanoDSF and Differential Scanning Calorimetry DSC based on the thermal unfolding of lysozyme is presented. The unfolding of proteins is a well-understood problem, widely studied by thermal analysis such as Differential Scanning Calorimetry DSC, which needs high amounts of protein together with large protein concentrations. On the other hand, Differential Scanning Fluorimetry nanoDSF offers the advantage of requiring only small quantities and volumes of protein. For the first time, this study presents an evaluation model of the degree of unfolding α obtained from the nanoDSF fluorescence signal in order to determine the thermodynamic parameters. The thermal unfolding of the model protein lysozyme was measured at varying ranges of pH values and compared with the literature. Subsequently, the evaluation protocol of the nanoDSF fluorescence signal was applied to study the thermal stability of HMGB1, a redox-sensitive protein involved in numerous biological functions. NanoDSF enabled the determination of thermodynamic parameters with minimal protein sample requirements. Here, the impact of the disulfide bridge between Cys23 and Cys45 on the stability of the HMGB1 protein was explored. Several methods, like Differential Scanning Fluorimetry nanoDSF, fluorescence spectroscopy FT-IR, circular dichroism CD, and high-pressure FT-IR, were employed to quantify the unfolding and folding transitions. To understand its possible contribution to the stability and flexibility of HMGB1. The variants of the HMGB1 protein A-Box, B-Box, (A+B)-Box and a full-length HMGB1 were analysed. Moreover, the stability and flexibility of HMGB1 for the different redox states of A-Box and full-length HMGB1 were proved. Those different thermodynamic fingerprints of HMGB1 appear to have biological consequences, defining its function, as the redox state is closely connected to it and depends on the surrounding environment of the protein. Depending on its redox state, the HMGB1 protein changes the conformation and has distinct roles in the organism.