Biophysical Characterization of the Golgi Reassembly and Stacking protein (GRASP) from Trypanossoma cruzi

The Golgi complex (GC) is part of the endomembrane system in eukaryotic cells. Although not much is known about the mechanisms that define its cellular shape, it is understood that the structure and function of the Golgi complex are closely correlated. The organization and function of the Golgi require Golgi matrix proteins that assist in connecting the cisternae and secretory vesicles. One of the matrix proteins that serve this function is Golgi reassembly and stacking protein (GRASP), which is also involved in unconventional protein secretion mechanisms. The flagellated protozoan Trypanossoma cruzi possesses organelles similar to eukaryotic cells, including the Golgi complex. Currently, no structural data are available on the GRASP protein of Tripanossoma cruzi, making this project groundbreaking as it addresses heterologous expression, purification, and characterization of that GRASP protein in solution. Our comprehensive study aims to unveil structural features through biochemical and biophysical characterizations. We successfully expressed and purified TcGRASP, which showed excellent yield and purity. Circular Dichroism (CD) suggests proper folding post-purification, with a predominance of disordered regions and alpha helices in the secondary structure. We assessed denaturation using ellipticity at 222 nm, revealing that the process followed a sigmoid-like, cooperative unfolding curve with a half-transition concentration of approximately 4.2 M urea. The protein exhibited remarkable thermal stability, with a mid-transition temperature (TM) of approximately 56oC as determined via CD and DSC. We obtained structural data of the protein in solution, which can lead to a better understanding of its participation during the life cycle of the Trypanosomatidae family parasites. This could potentially aid in developing drugs to treat Chagas disease.