Propensity of GRASPs to Undergo Liquid-Liquid Phase Separation Studied by an Integrated SAXS.

Liquid-liquid phase separation (LLPS) is an important mechanism for the formation of biomolecular condensates and plays a central role in cellular organization. Golgi Reassembly and Stacking Proteins (GRASPs) are intrinsically disordered proteins involved in Golgi organization and protein secretion, and their propensity to undergo LLPS may provide new insights into their biological functions and non conventional signaling mechanisms. Previous studies have demonstrated that some GRASPs can undergo concentration- and environment-dependent self-association/phase separation. In this work we aim to investigate ancestral GRASPs in different  physicochemical conditions including variation in protein concentration, pH, ionic strength, temperature and presence or absence of polyethylene glycol (PEG). Preliminary SDS-PAGE analysis showed a predominant band at approximately 22 kDa, consistent with the expected molecular mass of the ancestral GRASP. Further experiments are being performed to determine how physicochemical conditions modulate its structural organization and phase behavior. SAXS and XPCS will be performed at Sirius Sapucaia beamline and the results will be compared with other results from human GRASPs and yeast GRASPs. These complementary techniques will enable the characterization of structural changes and molecular dynamics during protein aggregation and condensate formation, from the nanoscale to the micrometer scale and from milliseconds to minutes.