Oxidative refolding and NMR analysis of the second extracellular loop of human Occludin: The C216-C237 disulfide bond as a structural linchpin.

Occludin (OCLN) is a key integral membrane protein of tight junctions, essential for maintaining the blood-tissue barrier and serving as a critical co-receptor for Hepatitis C Virus entry. Its second extracellular loop (EL2) is a primary determinant of junctional permeability and viral tropism, yet its structural characterization has been hampered by the technical challenges of achieving native oxidative folding in vitro. In this study, we established a robust platform for the recombinant expression and purification of the human OCLN EL2 domain. By optimizing affinity tag placement and developing an efficient oxidative refolding protocol followed by RP-HPLC purification, we obtained a highly pure, folded EL2 domain. Circular dichroism spectroscopy revealed a structured conformation with significant helical content, while 1H-15N HSQC NMR spectroscopy provided atomic-resolution evidence of a stable tertiary fold. Notably, this fold is strictly dependent on the conserved C216-C237 intramolecular disulfide bond. Chemical reduction of this single covalent bridge resulted in a global collapse of the structural dispersion and a transition to a disordered state, as evidenced by narrowed NMR linewidths and loss of spectral complexity. These findings substantiate the role of the OCLN EL2 domain as a structural redox sensor and provide a reliable methodology for producing high-quality samples for future high-resolution structural studies and the development of targeted biotherapeutics.