Metallobiological necklaces: mass spectrometric and molecular modeling study of metallation in concatenated domains of metallothionein.

The ubiquitous protein metallothionein (MT) has proven to be a major player not only in the homeostasis of Cu(I) and Zn(II), but also binds all the Group 11 and 12 metals. Metallothioneins are characterised by the presence of numerous cys-x-cys and cys-cys motifs in the sequence and are found naturally with either one domain or two, linked, metal-binding domains. The use of chains of these metal-thiolate domains offers the possibility of creating chemically tuneable and, therefore, chemically dependent electrochemical or photochemical surface modifiers or as nanomachinery with nanomechanical properties. In this work, the metal-binding properties of the Cd(4)-containing domain of alpha-rhMT1a assembled into chains of two and three concatenated domains, that is, "necklaces", have been studied by spectrometric techniques, and the interactions within the structures modelled and interpreted by using molecular dynamics. These chains are metallated with 4, 8 or 12 Cd(II) ions to the 11, 22, and 33 cysteinyl sulfur atoms in the alpha-rhMT1a, alphaalpha-rhMT1a, and alphaalphaalpha-rhMT1a proteins, respectively. The effect of pH on the folding of each protein was studied by ESI-MS and optical spectroscopy. MM3/MD simulations were carried out over a period of up to 500 ps by using force-field parameters based on the reported structural data. These calculations provide novel information about the motion of the clustered metallated, partially demetallated, and metal-free peptide chains, with special interest in the region of the metal-binding site. The MD energy/time trajectory conformations show for the first time the flexibility of the metal-sulfur clusters and the bound amino acid chains. We report unexpected and very different sizes for the metallated and demetallated proteins from the combination of experimental data, with molecular dynamics simulations.