HUMAN GOLGIN-45 UNDER THE LENS: NOVEL BIOPHYSICAL INSIGHTS INTO STRUCTURE AND COMPLEX FORMATION WITH GRASP55

The Golgi Complex is a central organelle in eukaryotic cells, responsible for the trafficking, processing, and sorting of proteins and lipids. The maintenance of its unique structure is crucial for Golgi function and relies on a group of proteins known as Golgi Matrix Proteins, consisting mainly of Golgi Reassembly and Stacking Proteins (GRASPs) and Golgins. Golgins are a family of proteins predicted to be formed by extended coiled coils, with a predicted length between 100-600 nm. They are involved in vesicular traffic, Golgi maintenance, dynamics, and positioning within cells. Golgins associate with the Golgi cisternae via their carboxyl terminus, interacting directly with the membrane surface or through a membrane-associated partner. Despite the importance, there are no studies on the structural and biophysical characterization of members of the Golgin family, even fundamental aspects such as the actual existence of coiled-coil domains have not yet been proven, and are completely based on computational predictions of domains. Considering the gap in knowledge of the structural properties of Golgins, the present study focuses on one of these members, specifically human Golgin-45. An efficient purification protocol for Golgin-45 protein was successfully established, allowing for its detailed structural characterization. Circular Dichroism thermal denaturation assays revealed a structural change in Golgin-45, transitioning from an alpha-helix configuration to a beta-sheet-rich structure. Dynamic Light Scattering experiments at varying protein concentrations indicated that Golgin-45 undergoes concentration-dependent oligomerization. In addition, this study aims to investigate the molecular interaction between Golgina-45 and its partner GRASP55. Results obtained by SEC-MALS confirm the formation of a stable protein complex, with a molecular mass compatible with a heterotetramer, possibly consisting of two subunits of each protein. These data provide novel insights into the conformational dynamics and oligomerization behavior of Golgin-45, as well as its interaction with GRASP55.