Zn-metallothionein complexes studied by Raman spectroscopy.

Metallothioneins (MTs) are low molecular weight, cysteine-rich proteins, with an exceptional heavy metal coordination capacity. These metal chelating peptides play an active role in zinc homeostasis. Although their high heterogeneity, structural and functional studies have been mainly devoted to vertebrate and fungal MTs. Participation of metal ligands other than Cys and the presence of secondary structure elements in metal-MT complexes are fairly unknown, especially in non-vertebrate MTs. Six in vivo-synthesized Zn-MTs, representative of different MT families (mollusc, insect, nematode, echinoderm, plant and vertebrate) were studied by analytic and spectroscopic techniques. The examined MTs contain from 43 to 73 amino acids, among which at least a 30% are Cys, whereas very few are aromatic residues. Zn(II)-MT complexes were heterologously synthesized in E.coli, to obtain aggregates representative of those formed in biological systems. These complexes contain variable amounts of metal and sulfide ions, quantitatively evaluated by analytical measurements. The formation of more than one species (S2-containing and S2-devoid complexes), revealed by ESI-MS spectra, is a constant for all these MTs. The analysis of the Raman spectra gave information about the secondary structure of the metal-MT aggregates. For all the MT examined, a relevant contribution of beta-turns was shown, whereas in all cases the alfa-helix content resulted almost negligible. As regards Cys sulfurs, almost all Cys residues present in MTs resulted to be involved in the metal coordination, as indicated by the presence of several bands attributable to the metal-S stretching modes at low wavenumbers (< 500 cm-1). The high number of vM-S bands and their broadening suggest the formation of different metal centres. Significantly, Raman bands useful as markers of sulfide bridging ligands were identified. In Zn-CeMT2 (from CElegans) and Zn-QsMT (from plant - Quercus suber) the eventual participation of the His residue in metal binding was evaluated through a curve fitting analysis of the 1630-1565 cm-1 Raman spectral range. His residues resulted to be mainly coordinated in Zn-CeMT2 (~ 90%), whereas in Zn-QsMT His is mainly present as free tautomer (~ 90%). Raman spectroscopy showed to be a powerful technique, able to provide information on the state of the Cys sulfur (metal coordinated and oxidised), the metal binding environment (i.e. the participation of His in metal coordination), as well as on the secondary structure.