Studies of metal binding reactions in metallothioneins by spectroscopic, molecular biology, and molecular modeling techniques

Abstract

Metallothioneins (MTs) are a class of metal-binding proteins characterized by a high cysteine content (up to 30% of the amino acid residues), low molecular weight, and lack of aromatic amino acid residues. Remarkable metal-binding properties have been reported both in vivo and in vitro by a number of different analytical techniques. Chemical and spectroscopic studies have shown that an unusually wide range of metals bind to MT. 113Cd and 1H-NMR techniques have been used successfully to determine the structures in a number of different proteins. Together with X-ray diffraction results, analyses of these NMR data have established that in mammalian Cd 7–MT and Zn 7–MT, the metals are tetrahedrally coordinated in two isolated domains with stoichiometries of M 4S 11 and M 3S 9. Recently, Stillman, collaborators, and coworkers have characterized the Zn(II), Cd(II), Cu(I), Ag(I), Au(I) and Hg(II) containing forms of MTs from mammalian, yeast, and recombinant human sources using a number of different spectroscopic techniques. Optical spectroscopy, and in particular circular dichroism and luminescence, have provided rich details of a complicated metal binding chemistry, whether metals are added directly to the metal free, apo-MT, or to the Zn-containing protein. Electrospray ionization mass spectrometry is a powerful technique for the characterization of proteins in general directly from solution. For MTs, both from mammalian and recombinant sources, use of this technique allows study of the details of metal binding to the protein as a function of metal loading and pH. Kinetic studies allow determination of the metal binding mechanism when Cu(I) binds to both the metal-free and Zn(II) protein. Scanning tunneling microscopy is a surface analysis technique capable of producing nanoscale images with atomic resolution and is being applied to obtain images of MT on Au(111) surfaces. Molecular modeling, together with XANES, XAFS, and structural information from 2D- 1H-NMR data allow the determination of 3D structures of a range of MTs. Finally, molecular dynamics (MD) calculations have been carried out to investigate the motion of the metal-containing and metal free peptides, with special interest in the region of the metal binding site.