Role of aqueous two-phase systems in modulating the kinetics and dynamics of biological processes

Abstract

Liquid-liquid phase separation (LLPS) phenomena have contributed immensely to the field of cell biophysics and cell biology in providing rational answers to the process of formation of membraneless organelles. These membraneless organelles play a vital role in understanding the origin of life in terms of cellular compartmentalisation. An increasing number of proteins and protein-nucleic acid mixtures has been shown to undergo LLPS at high concentrations forming two liquid phases - a protein-rich phase and a phase with diluted protein concentration. The tendency of LLPS to concentrate proteins comes with an added danger of aggregation, however, which is an issue in many human diseases such as amyloid formation in Alzheimer's disease or synuclein plaques in Parkinson's disease. High Hydrostatic Pressure (HHP) is a sophisticated tool to yield novel information on the free-energy and conformational landscape of biomolecules. Further, Earth being a predominantly high-pressure region makes it imperative to use this tool to understand the adaptability of piezophiles. However, the combined effect of LLPS and HHP on biological reactions and cellular processes, such as enzyme kinetics and ligand binding, has yet not been experimentally determined. A part of the thesis explores the combined effects of an aqueous two-phase system (ATPS) formed by the synthetic polymers polyethylene glycol (PEG) and dextran invoking LLPS and pressure on the enzymatic hydrolysis reaction of the substrate AAF-AMC catalysed by the proteolytic enzyme α-CT. In general, ATPS affects the enzymatic reactions in a variety of ways, such as partitioning in different or the same phases, increasing the effective concentration of solutes and thus inducing an excluded volume effect. Pressure, on the other hand, can increase the reaction rate and, alter the substrate specificity and stereoselectivity of an enzyme by choosing the product with a smaller partial volume, increases conformational flexibility. In this work it was observed that HHP did not have any marked effect on the kinetic constants in the ATPS, which was not possible to explain by simple steric crowding. Additional contributions, such as changes in water activity and non-specific weak interactions with ATPS components have to be considered to explain the results obtained. These results are important for understanding the use of LLPS and ATPS in modulating an enzymatic reaction for biotechnological use. The other part of this thesis focusses on the combined effects of pressure and ATPS on a ligand binding reaction, i.e., on the binding phenomenon between Bovine Serum Albumin (BSA) and 1-anilino-8-naphthalene (ANS) which was monitored through fluorescence spectroscopy. The results indicate that unlike buffer where the binding affinities to the three equivalent and independent binding sites of BSA could not be distinguished, the presence of ATPS gives rise to two binding modes, thus allowing to differentiate between weak and strong binding of the three sites. This result can be explained by considering soft interactions between the binding sites on BSA with the crowder molecules, such as PEG and dextran. Upon pressurization, the binding affinities of all binding sites decrease, which can be due to the unfavourable effect of pressure on hydrophobic interactions as well as a volume change due to release of water molecules and creation of void volume upon binding. These results also demonstrate that pressure dependent studies are able to reveal differences in binding sites of ligands, owing to the high accuracy by which volume changes can be determined.