Material Properties of Neutral Globular Protein-Based Condensates

In the world of cellular biology, the study of membrane-less organelles has recently gained significant traction, revealing a complex and dynamic world of cellular organization. These organelles are formed through phase separation, where biomolecules transition from an originally homogeneous solution to a solution containing a distinct dilute phase and a dense phase, termed a biomolecular condensate. Complex coacervation, involving the phase separation of oppositely charged polyelectrolytes, has been a focal point in exploring these phenomena, often highlighting the role of electrostatic interactions in protein aggregation and the formation of condensates.

We have previously concentrated on the interactions between positively charged green fluorescent proteins (GFP), and negatively charged biomolecules. While these studies provide valuable insights, they have predominantly emphasized electrostatic forces. However, it is becoming increasingly clear that non-electrostatic interactions, such as hydrophobicity, π-π stacking, and van der Waals forces, also play a crucial role in the formation and stabilization of biomolecular condensates.

In this study, we address this gap by engineering a library of eight neutral GFPs, each tagged with short peptides containing a few aromatic residues, to investigate the non-electrostatic mechanisms that drive phase separation in cellular contexts. By utilizing Escherichia coli as a model system, we can analyze how these neutral proteins phase separate to form condensates, focusing particularly on the influence of aromatic interactions. This approach allows us to dissect the contribution of specific amino acid properties and patterning to the overall material characteristics of the condensates formed.

Preliminary findings suggest that the pairing of amino acids like phenylalanine, tryptophan, and tyrosine enhances phase separation through increased hydrophobic interactions and π-π stacking, resulting in more stable condensate structures. However, it remains unclear whether the observed biomolecular condensates are formed through protein-RNA interactions or self-protein interactions known as simple coacervation. On-going work, both in vivo and in vitro works on probing the conditions under which these proteins condense and identifying the type of interactions leading to condensation. With this foundational understanding, we can begin to build biomolecular condensates with more complex, and biologically relevant, structures such as multiphase, multi-layered condensates.