BIOPHYSICAL CHARACTERIZATION OF THE GOLGI REASSEMBLY AND STACKING PROTEIN (GRASP) FROM PLASMODIUM FALCIPARUM

Grasps are a family of peripheral membrane proteins found in the Golgi complex. GRASP proteins play a role in Golgi ribbon formation and, more recently, they were found as key molecules in unconventional protein secretion (UPS) pathways. Their N-terminal region contains two PDZ domains, which are tandemly connected, exhibiting a unique promiscuity of interactions with various partner proteins and an uncommon structural plasticity. The C-terminal region of GRASPs displays a regulatory functional character that is not conserved across species or even among paralogous genes. It is enriched in proline, serine, and threonine residues (SPR domain) and possesses unstructured regions. Studies suggest that GRASPs function through dimerization and membrane anchoring via direct interactions with Golgins, along with covalent modification, like myristoylation, at Glycine-2 residue. Furthermore, in vitro assays have demonstrated the phase separation of GRASPs, as reported by our research group. The parasite Plasmodium falciparum expresses two GRASP isoforms, PfGRASP1 and PfGRASP2. PfGRASP1 has a myristoylation motif, similar to vertebrate GRASPs. PfGRASP2 has its N-terminal region characterized by an amphipathic helix and acetylation, both contributing to membrane interactions, resembling fungal GRASPs. Notably, P. falciparum exhibits a simple and "unstacked" Golgi architecture, raising the question of why a second GRASP protein has evolved. To address this issue, our objective was to perform the biophysical characterization of PfGRASP1 from P. falciparum, establishing suitable expression and purification protocols for subsequent fluorescence, circular dichroism and differential scanning calorimetry measurements, to enhance our understanding of the protein's structural features. Additionally, we aimed to deduce conditions capable of inducing in vitro phase separation of the protein, with the purpose of evaluating whether phase separation is a pattern of GRASPs. These findings are important for future studies on the biological functions and structural properties of PfGRASP1, which may contribute to advancing scientific knowledge in this field.