Abstract
Here, we demonstrate that a metal ion binding motif could serve as an efficient and robust tool for site-specific conjugation strategy. Cysteine-containing metal binding motifs were constructed as single repeat or tandem repeat peptides and their metal binding characteristics were investigated. The tandem repeats of the Cysteine-Glycine-Histidine (CGH) metal ion binding motif exhibited concerted binding to Co(II) ions, suggesting that conformational transition of peptide was triggered by the sequential metal ion binding. Evaluation of the free thiol content after reduction by reducing reagent showed that metal-ion binding elicited strong retardation of cysteine oxidation in the order of Zn(II)>Ni(II)>Co(II). The CGH metal ion binding motif was then introduced to the C-terminus of antibody heavy chain and the metal ion-dependent characteristics of oxidation kinetics were investigated. As in the case of peptides, CGH-motif-introduced antibody exhibited strong dependence on metal ion binding to protect against oxidation. Zn(II)-saturated antibody with tandem repeat of CGH motif retains the cysteine reactivity as long as 22 hour even with saturating O2 condition. Metal-ion dependent fluorophore labeling clearly indicated that metal binding motifs could be employed as an efficient tool for site-specific conjugation. Whereas Trastuzumab without a metal ion binding site exhibited site-nonspecific dye conjugation, Zn(II) ion binding to antibody with a tandem repeat of CGH motif showed that fluorophores were site-specifically conjugated to the heavy chain of antibody. We believe that this strong metal ion dependence on oxidation protection and the resulting site-selective conjugation could be exploited further to develop a highly site-specific conjugation strategy for proteins that contain multiple intrinsic cysteine residues, including monoclonal antibodies.
Highlights
Site-selective conjugation at proteins has been widely employed to detect cellular distribution, molecular interactions, cellular trafficking, and changes in mobility or conformation of proteins [1,2,3,4,5]
N-terminal acetylation removes the complexity associated with pH-dependent changes in the coordination environment of the CGH motif and reflects the possible amino acid environment when it is introduced into a protein
When we introduced a serine mutation replacing cysteine, the Co(II) binding capability was abrogated, indicating that thiol groups of cysteine are critical to the metal binding pocket in the CGH motif
Summary
Site-selective conjugation at proteins has been widely employed to detect cellular distribution, molecular interactions, cellular trafficking, and changes in mobility or conformation of proteins [1,2,3,4,5]. Many researchers have developed sophisticated conjugation technologies to label.
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