title: Conformational Study of an Artificial Metal-Dependent Regulation Site for Use in Designer Proteins.
abstract: 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 resultingbipy-GS2andterpy-GS2were investigated as potential metal ion dependent switches in aqueous solution, and were found to predominantly adopt thetransoïdconformation at physiological pH. Metal complexation with CuIIand ZnIIat this pH has been studied by UV/Vis, CD, NMR and ion-mobility mass spectrometry. ZnIItitrations are consistent with the formation of a 1:1 ZnII:terpy-GS2complex at pH 7.4, butbipy-GS2was 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 logKM6.86 (bipy-GS2) and 6.22 (terpy-GS2), consistent with a higher affinity for the unconstrained bipyridine, compared to the strained terpyridine. CuIIcoordination involves the initial formation of 1:1 complexes, followed by 1.5Cu:1bipy-GS2and 2Cu:1terpy-GS2complexes at micromolar concentrations. Binding constants for formation of the 1:1 complexes (logKM12.5 (bipy-GS2); 8.04 and 7.14 (terpy-GS2)) indicate a higher affinity for CuIIthan ZnII. 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.
authors.affiliation: School of Chemistry, University of Birmingham Edgbaston, B15 2TT, UK.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Oheix
authors.affiliation: School of Chemistry, University of Birmingham Edgbaston, B15 2TT, UK.
authors.forename: Neil
authors.initials: N
authors.lastname: Spencer
authors.affiliation: Waters Corporation Atlas Park, Simonsway, Wythenshawe, Manchester, M22 5PP, UK.
authors.forename: Lee A
authors.initials: LA
authors.lastname: Gethings
authors.affiliation: School of Chemistry, University of Birmingham Edgbaston, B15 2TT, UK.
authors.forename: Anna F A
authors.initials: AF
authors.lastname: Peacock
keywords: Bioinorganic chemistry; Bipyridine; Metalloswitches; Peptides; Terpyridine