Biomolecular condensates (BMCs) are naturally occurring membraneless organelles formed through liquid–liquid phase separation (LLPS). They play essential roles in cellular processes such as signal transduction, gene expression, and stress response. Although LLPS involving intrinsically disordered proteins (IDPs) and intrinsically disordered protein regions (IDPRs) is well documented, far less is known about LLPS in folded globular proteins. Recent studies indicate that folded globular proteins can also form crowder induced condensates; however, the factors that drive LLPS in these systems remain poorly understood. Moreover, the impact of LLPS on protein function is also understudied.We are examining how variables such as protein concentration, molecular crowding, pH, temperature, and ionic strength influence LLPS induced BMC formation. In addition, we are investigating how LLPS affects enzyme activity. Our study uses modular globular proteins, such as bovine serum albumin and Escherichia coli prolyl tRNA synthetase, alongside synthetic polymers like polyethylene glycols of varying sizes. To probe the mechanistic details of LLPS, we employ a suite of complementary techniques including fluorescence spectroscopy, confocal imaging, light scattering measurements, and enzymatic kinetic assays.
