Magnetic and Metal Binding Structural Analysis of Mn,Zn-Metallothionein-Green Fluorescence Fusion Protein

Metallothionein-green fluorescence fusion protein (MT-GFP) is a zinc binding protein, which binds to seven divalent transition metal ions through its 20 conserved cysteines and forms two metal binding clusters with Zinc-Blende structure. In this study, we substituted Mn2+ for Zn2+ at M3, M4 metal binding sites in the b-domain of MT-GFP. We found this Mn, Zn binding protein exhibited ferromagnetic properties from 10K to 300K by SQUID measurement. By micro-Raman spectroscopy analysis, the Zn-S and Mn-S bending modes can observed clearly at 288 cm−1 and 355 cm−1, respectively. These indicate that the Zn2+ and Mn2+ are bound with Cys residues of MT-GFP. Extended X-ray absorption fine structure (EXAFS) analysis also indicated Mn2+ binding to MT-GFP via the Mn-S bond of Cys.