INSIGHT INTO HUMAN TMED1 PROTEIN: EXPRESSION, PURIFICATION, AND CHARACTERIZATION

TMED proteins are eukaryotic transmembrane proteins located in all subcompartments of the secretory pathway: endoplasmic reticulum, Golgi, and the intermediate compartments, playing a vital role in the bidirectional transport between these organelles. However, crucial information regarding their structure, oligomeric state, and cargo anchoring mechanisms remains unknown. Our study focuses on a comprehensive biophysical analysis of the GOLD domain of human TMED 1. After purifying the recombinant TMED 1 GOLD protein, we obtained structural data using various techniques such as Circular Dichroism, Size Exclusion Chromatography with Multi-Angle Light Scattering, Differential Scanning Calorimetry, X-ray crystallography, Phylogenetic analyses, Molecular dynamics simulations, and MM/PBSA free energy calculation. Protein crystallography revealed a high-resolution structure, diffraction to 1.72 Å resolution, with a β-sandwich organization comprising two four-strand antiparallel β-sheets and a conserved disulfide bond. This finding aligned with previous data, indicating the protein's dimeric nature, which was confirmed experimentally in solution. Molecular dynamics simulations further revealed entropic stabilization at approximately -28 Kcal.mol-1 and highlighted key residues involved in oligomerization. We proposed a dimer model orientation towards the lipid membrane based on the protein's surface electrostatic charge.