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5TH INTERNATIONAL CONFERENCE ON METALLOTHIONEIN SYMPOSIUM PAPERS

Peptide Folding, Metal-Binding Mechanisms, and Binding Site Structures in Metallothioneins

Department of Chemistry, The University of Western Ontario, London, Ontario, Canada N6A 5B7

To whom requests for reprints should be addressed at ¹ Department of Chemistry, The University of Western Ontario, London, ON, Canada, N6A 5B7. E-mail: martin.stillman{at}uwo.ca

Abstract

This minireview specifically focuses on recent studies carried out on structural aspects of metal-free metallothionein (MT), the mechanism of metal binding for copper and arsenic, structural studies using x-ray absorption spectroscopy and molecular mechanics modeling, and speciation studies of a novel cadmium and arsenic binding algal MT. Molecular mechanics–molecular dynamics calculations of apo-MT show that significant secondary structural features are retained by the polypeptide backbone upon sequential removal of the metal ions, which is stabilized by a possible H-bonding network. In addition, the cysteinyl sulfurs were shown to rotate from within the domain core, where they are found in the metallated state, to the exterior surface of the domain, suggesting an explanation for the rapid metallation reactions that were measured. Mixing Cu₆ß-MT with Cd₄-MT and Cu₆-MT with Cd₃ß-MT resulted in redistribution of the metal ions to mixed metal species in each domain; however, the Cu ⁺ ions preferentially coordinated to the ß domain in each case. Reaction of As^{3 +} with the individual metal-free ß and domains of MT resulted in three As^{3 +} ions coordinating to each of the domains, respectively, in a proposed distorted trigonal pyramid structure. Kinetic analysis provides parameters that allow simulation of the binding of each of the As^{3 +} ions. X-ray absorption spectroscopy provides detailed information about the coordination environment of the absorbing element. We have combined measurement of x-ray absorption near edge structure (XANES) and extended x-ray absorption fine structure (EXAFS) data with extensive molecular dynamics calculations to determine accurate metal-thiolate structures. Simulation of the XANES data provides a powerful technique for probing the coordination structures of metals in metalloproteins. The metal binding properties of an algal MT, Fucus vesiculosus, has been investigated by UV absorption and circular dichroism spectroscopy and electrospray ionization–mass spectrometry. The 16 cysteine residues of this algal MT were found to coordinate six Cd^{2 +} ions in two domains with stoichiometries of a novel Cd₃S₇ cluster and a ß-like Cd₃S₉ cluster.

Key Words: protein folding • MM/MD • Cd(II), Zn(II), Cu(I), As(III) • emission spectroscopy • metal domain • Fucus vesiculosus • kinetic analysis • EXAFS • XANES