From Protein Adsorption to Crystallization

Abstract

Proteins are essential to all forms of life. They are involved in almost all biological processes, acting as enzymes, structural components, transporting molecules, involved in signaling, and in immune responses. In addition, proteins are important in medicine, biotechnology, and industry. Many therapeutic drugs are based in proteins, and understanding protein function is essential for developing new treatments. Each protein has a specific function, which is determined by its unique three-dimensional structure.

Because the function of the protein is directly connected to its three-dimensional conformation, solving protein structure is essential. X-ray crystallography remains one of the most used methods for structural determination, but it requires high-quality protein crystals. Despite advances in protein crystallization research, growing suitable protein crystals is still a challenge. Protein crystallization is a complex process that depends on subtle balances between attractive and repulsive interactions. The crystallization mechanism for every known protein is still not understood. As a consequence, many proteins have not yet been crystallized, and the strategy is often based on trial and error, which is time-consuming.

This is why understanding the underlying mechanisms of protein crystallization is essential, and it is the main focus of this dissertation. Specifically, protein crystallization at a solid surface is often neglected, even though it is usually energetically favored compared to crystallization in the bulk solution. In this work, protein adsorption and crystallization at the interface are investigated using both surface-sensitive and bulk-sensitive techniques.

For this purpose, a suitable protein system was first identified. Two negatively charged globular proteins were screened under several conditions. beta-lactoglobulin (BLG) and human serum albumin (HSA) in the presence of three different trivalent salts: YCl3, LaCl3 and CeCl3. The main technique used in this work is a real-time surface-sensitive method called quartz crystal microbalance with dissipation monitoring (QCM-D), which is applied here for the first time to study protein crystallization at the surface. In the first results chapter, HSA in the presence of LaCl3 was chosen as the model system to study protein crystallization with QCM-D.

In the second results chapter, evidence for surface-assisted crystallization is presented. The crystallization of HSA in the presence of a trivalent salt was monitored in real-time. The experimental conditions were chosen near the phase boundary where protein-protein attractions are enhanced but amorphous aggregation and liquid-liquid phase separation (LLPS) are not present. A soft protein multilayer forms at the surface, which acts as a reservoir and environment for heterogeneous nucleation. As a result, protein crystals were only growing at the surface.

In the third results chapter, the same protein system was investigated with two different solvents. Heavy water (D2O) was compared to normal water (H2O). This substitution enabled a fine-tuning of protein interactions, providing a deeper understanding of the mechanisms behind protein crystallization at the surface. The results reinforce the connection between bulk properties and interfacial behavior, highlighting that small changes in the solution environment directly influence surface-induced crystallization.

Dissertation ist gesperrt bis 31. Juli 2027 !