Conformational Study of an Artificial Metal-Dependent Regulation Site for Use in Designer Proteins.

This report describes the dimerisation of glutathione, and by extension, other cysteine-containing peptides or protein fragments, with a 5, 5'-disubstituted-2, 2'-bipyridine or 6, 6"-disubstituted-2, 2':6',2"-terpyridine unit. The resulting bipy-GS₂ and terpy-GS₂ were investigated as potential metal ion dependent switches in aqueous solution, and were found to predominantly adopt the transoïd conformation at physiological pH. Metal complexation with Cu^II and Zn^II at this pH has been studied by UV/Vis, CD, NMR and ion-mobility mass spectrometry. Zn^II titrations are consistent with the formation of a 1:1 Zn^II:terpy-GS₂ complex at pH 7.4, but bipy-GS₂ was shown to form both 1:1 and 1:2 complexes with the former being predominant under dilute micromolar conditions. Formation constants for the resulting 1:1 complexes were determined to be log K_M 6.86 (bipy-GS₂ ) and 6.22 (terpy-GS₂ ), consistent with a higher affinity for the unconstrained bipyridine, compared to the strained terpyridine. Cu^II coordination involves the initial formation of 1:1 complexes, followed by 1.5Cu:1bipy-GS₂ and 2Cu:1terpy-GS₂ complexes at micromolar concentrations. Binding constants for formation of the 1:1 complexes (log K_M 12.5 (bipy-GS₂ ); 8.04 and 7.14 (terpy-GS₂ )) indicate a higher affinity for Cu^II than Zn^II. Finally, ion-mobility MS studies detected the free ligands in their protonated form, and were consistent with the formation of two different Cu adducts with different conformations in the gas-phase. We illustrate that the bipyridine and terpyridine dimerisation units can behave like conformational switches in response to Cu/Zn complexation, and propose that in future these can be employed in synthetic biology with larger peptide or protein fragments, to control large scale folding and related biological function.