Propensity of GRASPs to undergo liquid-liquid phase separation analyzed through an integrated approach of SAXS and XPCS

Golgi reassembly and stacking proteins (GRASPs) are members of the Golgi matrix protein family and play important roles in Golgi organization as well as in unconventional protein secretion under stress-related conditions. Increasing evidence suggests that these proteins undergo liquid-liquid phase separation (LLPS), yet the structural and dynamic mechanisms underlying their condensation remain poorly understood. Here, we propose to investigate phase separation in GRASP proteins using a synchrotron-based approach combining time-resolved small-angle X-ray scattering (SAXS) and X-ray photon correlation spectroscopy (XPCS). The combination of SAXS and XPCS offers a unique opportunity to correlate structural and dynamic information during condensate formation. SAXS will be used to track structural rearrangements, changes in assembly size, and nanoscale organization from early intermolecular association to the formation of condensed states. In parallel, XPCS will probe the dynamic behavior of the system by characterizing fluctuation modes, relaxation processes, and viscoelastic properties that emerge during phase separation. Together, these techniques will provide a multiscale description of the evolution of GRASP assemblies. Using this experimental framework, we will examine how intrinsically disordered regions contribute to the initial association of GRASP and how LLPS is modulated by temperature, PEG-induced crowding, and trivalent salts. The project also includes the development of optimized acquisition routines for time-resolved measurements, strategies to mitigate radiation damage, and data integration workflows combining SAXS and XPCS. Overall, this work aims to advance our understanding of LLPS in Golgi matrix proteins while establishing an integrated synchrotron-based platform for the structural and dynamic investigation of biomolecular condensates.