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

Schrimpf, Andreas, Hempel, Franziska, Li, Aitao, Linne, Uwe, Maier, Uwe G., Reetz, Manfred T., Geyer, Armin
Biochemistry 2018 v.57 no.26 pp. 3658-3664
Bacillariophyceae, dimerization, epoxide hydrolase, limonene, mass spectrometry, mixing, monoclonal antibodies, nuclear magnetic resonance spectroscopy, peptides, proteins, recombinant DNA, regioselectivity, thiols
Dimeric disulfide-linked peptides are formed by the regioselective oxidative folding of thiol precursors containing the CX₃CX₂CX₃C 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.