Interactions between proteins and ligands, which are fundamental to many biochemical processes essential to life, are mostly studied at dilute buffer conditions. The effects of the highly crowded nature of biological cells and the effects of liquid-liquid phase separation inducing biomolecular droplet formation as a means of membrane-less compartmentalization have been largely neglected in protein binding studies. We investigated the binding of a small ligand (ANS) to one of the most

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... onal proteins, bovine serum albumin (BSA) in an aqueous two-phase system (ATPS) composed of PEG and Dextran. Also, aiming to shed more light on differences in binding mode compared to the neat buffer data, we examined the effect of high hydrostatic pressure (HHP) on the binding process. We observe a marked effect of the ATPS on the binding characteristics of BSA. Not only the binding constants change in the ATPS system, but also the integrity of binding sites is partially lost, which is most likely due to soft enthalpic interactions of the BSA with components in the dense droplet phase of the ATPS. Using pressure modulation, differences in binding sites could be unravelled by their different volumetric and hydration properties. Regarding the vital biological relevance of the study, we notice that extreme biological environments, such as HHP, can markedly affect the binding characteristics of proteins. Hence, organisms experiencing high-pressure stress in the deep sea need to finely adjust the volume changes of their biochemical reactions in cellulo.