Résumé
High pressure (HP) is a particularly powerful tool to study protein folding/unfolding, revealing subtle structural rearrangements. Bovine I3-lactoglobulin (BLG), a protein of interest in food science, exhibits a strong propensity to bind various bioactive molecules. We probed the effects of the binding of biliverdin (BV), a tetrapyrrole linear chromophore, on the stability of BLG under pressure, by combining in situ HP small-angle neutron scattering (SANS) and HP-UV absorption spectroscopy. Although BV induces a slight destabilization of BLG during HP-induced unfolding, a ligand excess strongly prevents BLG oligomerization. Moreover, at SANS resolution, an excess of BV induces the complete recovery of the protein "native"3D structure after HP removal, despite the presence of the BV covalently bound adduct. Mass spectrometry highlights the crucial role of cysteine residues in the competitive and protective effects of BV during pressure denaturation of BLG through SH/S-S exchange. SIGNIFICANCE Pressure is a particularly sensitive tool for unfolding proteins and thus better understanding their folding pathway. Bovine I3-lactoglobulin (BLG), a protein of interest in food science, has a high propensity to bind various bioactive molecules. We show that biliverdin (BV) completely inhibits BLG oligomerization, making the pressure-induced unfolding of BLG completely reversible. We demonstrated the crucial role of binding to BLG cysteine residues in these protective effects of BV and proposed a disulfide bond exchange mechanism. Added to previous results, this study highlights the effects of various ligands on BLG under pressure as a function of their binding site, their affinity, and their chemical reactivity. Our strategy opens up new possibilities for the structural determination of protein intermediates and oligomers.