COMPARATIVE CONFORMATIONAL ANALYSIS OF TWO EVOLUTIONARILY DISTANT GRASP PROTEINS

Golgi Reassembly and Stacking Proteins (GRASPs) are evolutionarily conserved proteins involved in Golgi organization and unconventional protein secretion (UPS), highlighting their remarkable structural and functional versatility. Although structural information is available for mammalian GRASP domains, the conformational properties of full-length GRASPs from evolutionarily distant organisms remain poorly understood. Here, we present a comparative analysis of the sequence and structural properties of the recombinant GRASP proteins from D. discoideum (GrpA) and from T. cruzi (TcGRASP). Both proteins were heterologously expressed in E. coli and purified by Ni²⁺ affinity chromatography followed by size-exclusion chromatography (SEC). Purified fractions were analyzed by SDS-PAGE and circular dichroism (CD). Sequence alignment and disorder predictions were performed using Clustal Omega, IUPred2, and ANCHOR2. SDS-PAGE confirmed enrichment of the recombinant proteins after purification. GrpA displayed two well-resolved SEC peaks, whereas TcGRASP exhibited three distinct peaks, indicating a higher degree of conformational and/or oligomeric heterogeneity in solution. Sequence alignment revealed 22.5% global identity between TcGRASP and GrpA, which increased to 32.2% within the N-terminal PDZ core, while the C-terminal regions were markedly divergent. Disorder predictions indicated that both proteins contain extended flexible regions enriched in predicted interaction-prone motifs. TcGRASP displayed a broad CD minimum centered near 218 nm, consistent with significant β-sheet content together with a substantial contribution from flexible or disordered regions. In contrast, GrpA exhibited a CD spectrum dominated by a minimum near 200 nm and lacking well-defined α-helical features at lower temperatures, indicating a highly flexible and weakly ordered conformational ensemble. Temperature-dependent CD analyses revealed a progressive increase in the α-helical signal upon heating, suggesting partial temperature-induced structural ordering rather than the typical cooperative unfolding of globular proteins. These results show that, despite being homologous members of the GRASP family, TcGRASP and GrpA differ markedly in size, sequence composition, solution properties, and conformational behavior, highlighting the diversification of GRASP proteins in distantly related eukaryotes. This study establishes the foundation for the biophysical characterization of these non-mammalian GRASP proteins.