EXPLORING THE STRUCTURAL LANDSCAPE OF GRASP ISOFORMS IN Plasmodium falciparum

Plasmodium falciparum is the etiological agent of malaria and the primary cause of the severe clinical manifestations of the disease. Malaria remains a major global public health challenge. Golgi Reassembly and Stacking Proteins (GRASPs) constitute a family of Golgi membrane-associated proteins involved in unconventional protein secretion (UPS). The first GRASP family members identified in mammals were the isoforms GRASP55 and GRASP65. As research progressed, homologous proteins were characterized in several other eukaryotic organisms. Although most of these organisms possess only a single GRASP isoform, two isoforms, designated PfGRASP1 and PfGRASP2, have been identified in P. falciparum and are generated by alternative splicing of a single gene. All GRASPs comprise two domains: an evolutionarily conserved N-terminal GRASP domain containing two PDZ domains (Postsynaptic density protein 95, Discs large, and Zonula occludens-1) and a C-terminal intrinsically disordered region. This study aims to investigate the structural features and biophysical differences between the PDZ domains of the two isoforms to identify their potential functional implications. To this end, each PDZ domain was cloned into the pET22b expression vector and heterologously expressed in Escherichia coli Rosetta BL21(DE3) cells. Recombinant proteins were purified by Ni²⁺-NTA affinity chromatography using a column pre-equilibrated with HEPES buffer. The purified samples were then subjected to size-exclusion chromatography (SEC) using a Superdex 200 10/300 GL column. Particle size distribution was evaluated by mass photometry using a Refeyn TwoMP instrument, showing that they are mainly monomeric. The secondary structure contents of the PDZ domains were analyzed by circular dichroism (CD) spectroscopy, indicating differences in terms of helical content. Thermal stability and protein unfolding behavior were also investigated by CD thermal denaturation experiments over a temperature range of 20–90 °C. We will discuss the overall analysis of our data and pinpoint structural differences between the PDZ domains.