Complete 1H NMR sequential assignments have been made for copper(I)- and silver (I)-substituted metallothionein (MT) from Saccharomyces cerevisiae using standard 2D 1H NMR methods. The fingerprint region of the COSY spectrum of both metalloproteins shows a doubling of a few backbone proton resonances from residue K41 onward in the C terminus. This doubling of resonances is absent in the spectrum of the truncated mutant protein that lacks the five C-terminal residues which includes two cysteines. Concurrently, it has been established from a comparison of the heteronuclear 1H-109 Ag multiple-quantum coherence transfer (HMQC) spectrum on the silver-substituted mutant and the wild-type protein that metal ligation is similar in both molecules. Thus, the 2 C-terminal Cys are not essential for metal cluster formation in the wild-type yeast MT and only 10 of the 12 Cys present in this protein appear to be involved in ligating the 7 mol of bound metal ions. A qualitative analysis of the coupling constant, hydrogen exchange, and NOE data indicates the presence of many type I beta-turns and the lack of any other regular secondary structural elements. A comparison of chemical shifts and NOE data for native copper- and silver-substituted yeast MT indicates a high degree of conservation of structural elements in both proteins. Therefore, it seems reasonable to conclude that the metal to Cys connectivities which are obtained directly from the HMQC data on silver-substituted metallothionein are conserved in the native copper protein. Interestingly, a mixture of both 2 and 3 coordination was found for the bound Ag(I) ions in a single Ag7Cys10 cluster. This mixed coordination number and a single cluster arrangement is most probably also shared with the Cu(I) ion coordination in the native protein.

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Comparative Study | Journal Article | Research Support, Non-U.S. Gov't | Research Support, U.S. Gov't, Non-P.H.S. | Research Support, U.S. Gov't, P.H.S.

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Narula SS, Winge DR, Armitage IM

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