Role of Cosolutes on Lysozyme Diffusiophoresis and Condensation in Aqueous Mixtures

Abstract

Lysozyme is a model protein in physicochemical studies. This dissertation investigates two properties of lysozyme aqueous mixtures: salt-induced protein diffusiophoresis and liquid-liquid phase separation (LLPS). Diffusiophoresis is the migration of a particle induced by cosolute concentration gradients. This not-well-understood transport property is important for applications in separation science and microfluidics. In the dissertation first part, lysozyme diffusiophoresis induced by salt gradients was examined as a function of salt concentration at pH 4.5 and 25 °C for NaCl, KCl and MgCl2. Diffusiophoresis coefficients were extracted from multicomponent-diffusion data by applying non-equilibrium thermodynamics. A selected mass-transfer process was theoretically examined to show that MgCl2 concentration gradients produce significant lysozyme diffusiophoresis. Diffusiophoresis dependence on salt nature was theoretically examined and linked to protein charge. The effect of salt type on hydrogen-ion titration curves was characterized to understand role of salt on protein charge. Our findings indicate that MgCl2-induced protein diffusiophoresis can be exploited in protein separation science and adsorption-based biosensing. LLPS of protein aqueous mixtures is the reversible formation of protein-rich micro-droplets occurring below a well-defined temperature. This phase transition is metastable with respect to protein crystallization and aggregation. LLPS is important for protein crystallization and development of protein-based materials. It is also believed to be implicated in cell compartmentalization and protein-aggregation diseases. Lysozyme aqueous solutions reversibly undergo LLPS around 0 °C in the presence of additives such as NaCl near physiological composition. In the dissertation second part, it is shown that insertion of 4-(2-hydroxyethyl)-1-piperazineethanesulfonate (HEPES) as a second additive to lysozyme-NaCl-water mixtures reproducibly triggers conversion of protein-rich droplets into protein microparticles displaying crystalline nature. LLPS studies were extended to other additives sharing chemical similarities with HEPES. In these cases, droplets conversion into microparticles was either absent or drastically reduced. The phase diagram of the lysozyme-HEPES system was characterized. Measurements of lysozyme diffusion, HEPES supernatant concentration and heat of mixing show that lysozyme-HEPES interactions are weakly attractive and exothermic. Our findings indicate that additives with weak protein-ligand properties represent an important tool for controlling the fate of metastable protein-rich micro-droplets. This may be exploited in the preparation of protein-based materials and femtosecond crystallography.