Exploring Structural Aspects of the Human Golgi Matrix Protein GRASP55 in Solution

Abstract

AbstractIn mammalian cells, the Golgi apparatus is a central hub for intracellular trafficking, sorting and post-translational modifications of proteins and lipids. The Golgi reassembly and stacking proteins (GRASPs) are somehow involved in the Golgi stacking, which is significant for the proper function of the Golgi apparatus, and also in unconventional protein secretion. However, the structural details on how GRASPs accomplish those tasks are still elusive. In this context, we have explored the biochemical and biophysical properties of the human full-length GRASP55 in solution. Sequence-based analyses and circular dichroism spectroscopy suggest that GRASP55 presents multiple intrinsically disordered sites, although keeping considerable contents of secondary structure. Size exclusion chromatography coupled with multiple-angle light scattering (SEC-MALS) studies show that GRASP55 are monomers in solution. Urea denaturation of GRASP55 suggests that the transition to the unfolded state is a cooperative process. Differential scanning calorimetry (DSC) analysis displays two endothermic transitions for GRASP55, indicating the existence of an intermediate state prior to unfolding. Thioflavin T fluorescence shows that GRASP55 can form protein aggregates/fibrils at the intermediate state. Transmission electron microscopy and fluorescence lifetime imaging microscopy prove that GRASP55 forms large amorphous aggregates but not amyloid-like fibrils in the intermediate state. The significance of these results could be helpful in discussing the proper function of human GRASP55 in the Golgi organization as well as unconventional secretion of proteins.