CE06 - Polymères, composites, physique et chimie de la matière molle, procédés

Molecular forces and liquid-liquid separation in solutions of biological macromolecules – BioLLPS

Submission summary

Many membrane-less cellular compartments are formed by liquid-liquid phase separation (LLPS). This process is related to the flexibility and effective charge of macromolecules. The underlying mechanisms and driving forces are still poorly understood.

BioLLPS is based on a well-defined biological system used by the rabies virus to build viral factories in the infected host cell (called Negri bodies in the case of rabies). This system has unique properties for a biophysicist, as it separates into two liquid phases in a reversible and reproducible manner, and the highly concentrated protein phase remains in a liquid state without aggregation, crystallization, or gelation. This system also has the ability to control the partitioning of other macromolecules between phases. This system offers the possibility to be characterized in terms of structure and colloidal forces, allowing the identification of factors that control LLPS in biological systems.

The objective of BioLLPS is to decipher some of the underlying physicochemical principles by which proteins induce LLPS leading to membrane-free organelles in cells. In a bottom-up approach, we will establish a minimalist model system that recapitulates the essential properties of these virus-induced organelles: (1) appearance of a dense liquid phase and (2) partitioning of macromolecules.

Building on a series of preliminary results and observations, BioLLPS is organized into three work packages. WP1 aims to reconstitute LLPS from a purified protein that forms coacervates sensitive to temperature and osmotic pressure in vitro, and then establish the thermodynamics and kinetics of the resulting aqueous two-phase system (ATPS) as a function of osmotic pressure and salt concentration. We have a second protein that partitions into the dense phase of the previously obtained ATPS, and will use it to study its influence on the LLPS. We will develop a system that allows the rigorous control of the sample in particular by analytical ultracentrifugation. The objective of WP2 is to characterize the structural organization of proteins in the concentrated phase and to map the response of the ATPS to variations in electrolyte (salt) type and concentration and osmotic pressure (water activity) as a means of perturbing the molecular forces acting on the system. We will study the conditions of LLPS formation by light, X-ray and neutron scattering. WP3 aims at determining the mechanism of droplet formation by confronting the different existing models. We will test new theories of coacervation of synthetic polyelectrolytes, including two original elements: water activity and osmotic pressure difference due to Laplace pressure. The subject being at the frontier between viral biology and classical physical chemistry of colloids, our project is distinguished by the trans-disciplinary composition of the teams involved and will have impact in different scientific areas including fundamental biology, medical sciences, pharmacology and drug design.

The study of the link between LLPS and osmotic pressure could highlight the evolution of osmotic pressure in the infected cell as a temporal regulation mechanism of the viral cycle.

Project coordination

Marc Jamin (INSTITUT DE BIOLOGIE STRUCTURALE)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

ILL Institut Max von Laue - Paul Langevin / Large Scale Structures
IBS INSTITUT DE BIOLOGIE STRUCTURALE
ICSM Institut de Chimie Séparative de Marcoule

Help of the ANR 428,651 euros
Beginning and duration of the scientific project: - 48 Months

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