Liquid-Liquid Phase Separation of Supercharged Green Fluorescence Proteins in the Polyamine Biosynthetic Pathway Deleted E. coli.

Abstract

Protein liquid-liquid phase separation (LLPS) has attracted significant attention due to its involvement in cellular functions and has been believed to be the onset of neurodegenerative diseases. However, the primary mechanisms governing its formation and function remain unclear. Since most of proteins involved in condensation in cells are charged, it is hypothesized that charge-charge interactions mediated by small charged biomolecules, such as adenosine tri-phosphate (ATP) and polyamines, would be the primary driving force behind the protein LLPS. In the previous study, it is showed that small charged biomolecules are to be a main driver of protein LLPS through charge-charge interaction. Furthermore, multivalently charged small molecules play a crucial role in creating protein condensates when the protein themselves are charged, effectively acting as bridges between charged proteins. Having said that, the effect of endogenous polyamine molecules in protein condensation in cells could not be ruled out. In this study, an E. coli strain is utilized with a polyamine synthetase knocked-down, HT873, to investigate the role of polyamines in the aggregation of both negatively and positively charged green fluorescence proteins. This study suggests that both ATP and polyamine, as small charged biomolecules, play critical roles in the LLPS of charged proteins in cells.