Abstract:
Proteins are organic macromolecules consisting of a sequence that is composed of a series of amino acids (primary structure) joined together through peptide bonds. Their functionality is obtained through suitable folding (secondary structure) and assembly in three-dimensional space. Proteins are an essential component for ev- ery kind of life on earth. The functions of proteins are diverse and extensive. For example, enzymes catalyze chemical reactions which are of vitaly importance for metabolic processes of an organism. Other tasks include the transportation of various molecules as well as structure-forming functions such as the development of muscular tissue(s). Among other criteria, proteins can be divided into different subgroups based on their structural properties. This results in four subgroups, namely disordered proteins, fiber proteins, membrane proteins and globular pro- teins, the latter being especially relevant for this dissertation. The water-soluble globular proteins exhibit a rich and diverse phase behavior in the presence of polymers or polyvalent salts. The observed phenomena range from liquid-liquid phase separation (LLPS) to aggregation and crystallization. In addi- tion, “reentrant condensation” (RC) and “reentrant interaction” (RI) are particu- larly noteworthy. RC describes a phase behavior that is triggered by successively increasing a polyvalent salt concentration at a constant protein concentration. If a certain salt concentration is exceeded, aggregates or condensates form. A further increase beyond another defined salt concentration causes the previously formed aggregates or condensates to dissolve. The greater details of RI will be expanded upon in due course. Understanding and specifically manipulating the phase behavior of proteins is of fundamental importance for medicine, pharmacy and basic research. Protein crystals offer important information about the struc- ture of a protein, which in turn allows for conclusions regarding its function. This information would further our understanding to improve the understanding and potentially aid to treat unwanted aggregations or amyloid fibril formation, com- monly seen in Alzheimer’s disease patients. Chapter 7 addresses the effective interactions of four different globular proteins: ´- lactoglobulin, bovine serum albumin, human serum albumin and ovalbumin (BLG, BSA, HSA and OVA) admixed with the trivalent salt hexamine cobalt (III) chlo- ride (Hac). In previous studies, this salt has already been shown to induce a phase behavior known as reentrant condensation (RC) in Desoxyribonucleic acid (DNA) and Ribonucleic acid (RNA). This phase behavior is known to be an important concept in DNA packing and folding. Net negatively charged globular proteins such as BSA or HSA exhibit a similar phase behavior in the presence of trivalent salts such as YCl3 or LaCl3. The phase behavior is predominantly determined by a charge reversal, which is triggered by an increasing salt concentration at a constant protein concentration (cp). Within the scope of this work, it was then investigated whether the trivalent cobalt salt Hac can trigger the RC phase be- havior despite its structural differences to e.g. YCl3 or LaCl3. From a structural perspective, the Hac cation differs from other polyvalent metal cations by the fact that six ammonia ligands (NH3) are covalently bonded to the central cobalt atom (Co) thereby shielding the present (3+) charge. It transpired that the effective interactions induced by Hac are not sufficient to trigger RC. Instead, small-angle X-ray scattering experiments (SAXS) revealed that the BLG-Hac system induces a new phenomenon, called reentrant interaction (RI). Characteristically for pro- tein systems with RI, the SAXS profiles are similar to that of protein systems with RC phase behaviour; however, there is an absence of visible or measurable phase transitions occurring in RI. In parallel to the SAXS experiments, static and dynamic light scattering experiments (SLS, DLS) as well as visual investigations were carried out. Secondly (see Chapter 8), the effect of the polymer polyethylene glycol (PEG) on the protein systems BSA and HSA was investigated. Initially, a rich phase behavior was observed both visually and microscopically, which is characterized by liquid-liquid phase separation (LLPS), aggregate formation and, in the case of BSA, crystallization. The depletion effect, which is commonly known in the context of colloid theory, acts as the initiating effect. This unspecific, yet subtly equilibrium-based attractive interaction potential, which contrary to expectations is caused exclusively by repulsive interactions, can be manipulated and adjusted by tuning the polymer size and concentration. Systematic SAXS measurements of BSA and HSA-PEG systems in combination with quantitative modeling allow a better understanding of the protein phase behavior induced by the depletion ef- fect. Furthermore, the differences between the two related proteins are emphasized and further elaborated. Thus, HSA crystals offer a variety of different symmetry groups, whereas BSA has only been found to crystallize in the symmetry group C121. Furthermore, HSA can crystallize under unspecific (e.g. depletion effect) as well as specific (e.g. trivalent metal cations such as CeCl3) conditions, whereas BSA cannot. This is supported by surface analysis of the contact surfaces within a protein crystal. It transpired that HSA forms more contact surfaces and can therefore also crystallize in more, different symmetry groups compared to BSA. Chapter 9 examines the effects of temperature and effective interactions of BSA- LaCl3 solutions exposed to sodium-based Co salts (Na2SO4, NaCl, NaBr, NaNO3 and NaSCN) belonging to the Hofmeister series, alongside the phase behavior. The Hofmeister series divides anions into two groups: Cosmotropes and chaotropes. This classification is based on whether or not the anions percipitate proteins from a solution (cosmotropes SO42 – and chaotropes SCN – respectively). Similar to BSA-LaCl3, the BSA-LaCl3 co-salt systems exhibit a rich phase behavior which, in addition to RC, also features LLPS, as well as a lower critical solution tem- perature (LCST). The phase separation thus takes place during heating. Using temperature-controlled UV/vis measurements, it was shown that even the addi- tion of low concentrations (mM) of the Co salts to 80 mg/ml BSA with 10 mM- LaCl3 leads to a reduction of the LCST. The reduction of the LCST follows an inverse Hofmeister series, according to which chaotropic anions lower the LCST more strongly than cosmotropic anions. By approximating a Langmuir binding isotherm, it is possible to quantify and describe the association of the investigated anions with the protein surface more precisely. Complementarily performed sys- tematic SAXS experiments combined with model approximation provide further insights about the prevailing effective interactions, however, these only partially support the results from previous UV/vis measurements.
Small Angle X-ray Scattering