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bioRxiv · 10.1101/2025.03.29.646054

Cationic amino acid identity and net charge influence condensate properties in E. coli

Kidane, A. K.·Rosenfeld, J. R.·Johnston, J. D.·Dubbeldam, C.·Paraan, M.·Obermeyer, A. C.
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Abstract

Understanding the formation of biomolecular condensates (BMC) in biological systems has proven to be a paradigm shift in our understanding of the subcellular organization of biomacromolecules. From RNA metabolism, stress response mechanisms, and amyloidogenic pathologies, condensates have been implicated to play a role in a myriad of cellular phenomena. Despite their near ubiquity, we still do no wholly understand how the primary sequence of biomolecules influences their biophysical and rheological properties. Here, we aim to understand the impact of primary cationic amino acid composition on the properties of condensates. Using engineered recombinant proteins, we show that the formation and phase boundaries of coacervates formed between proteins and RNA is dependent on the cationic amino acid identity, as well as the net charge of the protein involved in condensation. Despite the equivalent charge between arginine and lysine at physiological pH, arginine has been shown to promote increased encapsulation efficiency and salt stability, as well as reduced protein mobility within condensates. We show that arginine-tagged globular proteins also have a higher salt resistance in vitro when compared to similar lysine-tagged globular proteins. This translates to a cellular context in which arginine tagged proteins promote increased condensate formation in model E. coli cells. We were also able to observe a reduction in the total fluorescent recovery and protein mobility within arginine-based condensates via FRAP. Together, these results suggest that in addition to electrostatic interactions and disorder as the main driving forces of phase separation in biological contexts, the primary sequence and side chain composition of proteins plays a significant role in dictating dynamics of coacervates.

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Kidane, A. K., Rosenfeld, J. R., Johnston, J. D., Dubbeldam, C., Paraan, M., Obermeyer, A. C.. 2025-04-03. Cationic amino acid identity and net charge influence condensate properties in E. coli. https://doi.org/10.1101/2025.03.29.646054

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