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Molecular dynamics study on the folding and metallation of the individual domains of metallothionein

De novo synthesis of metallothionein (MT) initially forms the metal‐free protein, which must, in a posttranslational reaction, coordinate metal ions via the cysteine sulfur ligands to form the fully folded protein structure. In this article, we use molecular dynamics (MD) and molecular mechanics (MM) to investigate the metal‐dependent folding steps of the individual domains of recombinant human metallothionein (MT). The divalent metals were removed sequentially from the metal‐sulfur M4(Scys)11 and M3(Scys)9 clusters within the α‐ and β‐ domains of MT, respectively, after protonation of the previously coordinating sulfurs. With each of the four (α) or three (β) sites defined, an order of metal release could be determined on the basis of a comparison of the strain energies for each combination by selecting the lowest energy demetallated conformations. The effect of an additional noninteracting, 34‐residue peptide sequence on the demetallation order was assessed when bound to either the N‐ or C‐termini of the individual domain fragments to identify the differences in cluster stability between one‐ and two‐domain proteins. The N‐terminal‐bound peptide had no effect on the order of metal removal; however, addition to the C‐terminus significantly altered the sequence. The number of hydrogen bonds was calculated for each energy‐minimized demetallated structure and was increased on metal removal, indicating a possible stabilization mechanism for the protein structure via a hydrogen‐bonding network. On complete demetallation, the cysteinyl sulfurs were shown to move to the exterior surface of the peptide chain. Proteins 2006. © 2005 Wiley‐Liss, Inc.