Walking Along a Protein Phase Diagram to Determine Coexistence Points by Static Light Scattering

Abstract

The physical process of liquid-liquid phase separation (LLPS), where the drive to minimize global free energy causes a solution to demix into dense and light phases, plays many important roles in biology. It is implicated in the formation of so-called "membraneless organelles" such as nucleoli, nuclear speckles, promyelocytic leukemia protein bodies, P bodies, and stress granules along with the formation of biomolecular condensates involved in transcription, signaling, and transport. Quantitative studies of LLPS in vivo are complicated by the out-of-equilibrium, multicomponent cellular environment. While in vitro experiments with purified biomolecules are inherently an oversimplification of the cellular milieu, they allow probing of the rich physical chemistry underlying phase separation. Critically, with the application of suitable models, the thermodynamics of equilibrium LLPS can inform on the nature of the intermolecular interactions that mediate it. These same interactions are likely to exist in out-of-equilibrium condensates within living cells. Phase diagrams map the coexistence points between dense and light phases and quantitatively describe LLPS by mapping the local minima of free energy versus biomolecule concentration. Here, we describe a light scattering method that allows one to measure coexistence points around a high-temperature critical region using sample volumes as low as 10μl.