PIONEERING GOLGIN PURIFICATION AND THE FIRST EXPERIMENTAL EVIDENCE OF COILED-COIL PRESENCE

The endoplasmic reticulum and the Golgi complex are essential organelles for the eukaryotic cell. They play a crucial role in the targeting, processing, and modification of newly synthesized proteins and  lipids, in the so-called classical secretory pathway. The Golgi displays a compartmentalized and polarized architecture, but also a dynamic structure due to the continuous flow of its components in the form of vesicles. Golgi matrix proteins, formed by the Golgi reassembly and stacking Proteins, and, most numerously, Golgins, act together to maintain the stacked structure of the cisternae and are essential for their ribbon-like organization in animals. Deciphering the mechanisms of these families of macromolecules will help us understand what guarantees the structure and function of the organelle. However, the complexity and dynamics of Golgins represent a challenge for researchers, who face difficulties in obtaining significant quantities of these proteins for detailed structural and biophysical studies. Expression in its native host was hindered by the intrinsically disordered nature of the protein, which rendered it highly susceptible to proteolytic degradation during cell lysis, further aggravated by heat generation from mechanical disruption methods such as probe sonication and French press. To overcome this, we established a novel expression system in Kluyveromyces lactis, allowing secretion of "Binder of USO1 and GRH1 protein 1", or BUG1, directly into the extracellular medium. This reduces exposure to cytoplasmic proteases and facilitates the recovery of intact protein for structural studies. We have also obtained robust experimental evidence confirming the presence of coiled-coil domains in BUG1, which had previously only been computationally predicted. Circular dichroism revealed typical α-helical spectra and high thermal stability. X-ray crystallography of the coiled-coil region confirmed a classic parallel dimer architecture. Calorimetric analysis further indicated intermediate folding states. Importantly, these findings are consistent with computational predictions developed in collaboration with Professor Fernando Melo, from the Institute of Biosciences, Letters and Exact Sciences (IBILCE), São José do Rio Preto campus, UNESP. These models, when compared to the crystallographic structure, reveal flexible regions that are the first to unfold during thermal transitions and enabling the formation of intermediate states.