Hinge-Type Dimerization of Proteins by a Tetracysteine Peptide of High Pairing Specificity.

Dimeric disulfide-linked peptides are formed by the regioselective oxidative folding of thiol precursors containing the CXCXCXC tetracysteine motif. Here， we investigate the general applicability of this peptide as a dimerization motif for different proteins. By recombinant DNA technology， the peptide CHWECRGCRLVC was loaded with proteins， and functional homodimers were obtained upon oxidative folding. Attached to the N-terminus of the dodecapeptide， the prokaryotic enzyme limonene epoxide hydrolase (LEH) completely forms a covalent antiparallel dimer. In a diatom expression system， the monoclonal antibody CL4 mAb is released in its functional form when its natural CPPC central parallel hinge is exchanged for the designed tetra-Cys hinge motif. To improve our understanding of the regioselectivity of tetra-disulfide formation， we provoked the formation of heterodimeric hinge peptides by mixing two different tetra-Cys peptides and characterizing the heterodimer by mass spectrometry and nuclear magnetic resonance spectroscopy.