The interior of a cell contains biomolecules of various sizes and chemical properties, impacting each other's structure and function. Generally, these molecules cause crowding by reducing the available space. Additionally, confinement occurs when the functional dynamics of biomolecules become severely compromised. A third consequence is liquid-liquid phase separation, when biomolecules cluster together, leading to phase separation within the intracellular environment. However, the molecular mechanism underlying these distinct phenomena is not fully understood. Using molecular dynamics simulation, we have been studying these phenomena by using synthetic polymer crowders and a multi-domain protein system - E. coli prolyl-tRNA synthetase. A member of the aminoacyl-tRNA synthetase family, this enzyme exhibits spectacular preorganizing dynamics of a distal domain that impacts its catalytic function. Three polymer crowders have been used: polyethylene glycols, Dextran 40k, and Ficoll 70k. Analysis of the interactions reveals different types of forces responsible for various protein-crowder assembly formations, which can explain the phase separation phenomenon. These theoretical observations are being validated using spectroscopic and microscopic tools.
