Research-carried-out

Amalia Muņoz

Research Interest

Postdoctoral work: Structure-Reactivity-Function Relatioships on Metallothionein

Study of the structure, chemical reactivity and biological function of mammalian and crustacean Metallothioneins. Small protein with an unusual high thiolate content that allows to bind up to 7 Cd²⁺or Zn²⁺ forming two independent clusters, alpha (S₁₁M₄) and beta (S₉M₃).

• Ligand exchange reactions of EDTA with Cd₇MT, Cd₄-alpha and Cd₃-beta (the isolated domains) was investigated and by following the release of Cd²⁺ bound to MT. The reaction showed to be biphasic for the holoprotein. The reaction mechanism proposed involves a fast removal of one Cd²⁺ from the alpha-domain, while the removal of the other metals involves a binding step that is EDTA-dependent with faster rate constants for the beta-domain.

• The possibility that each domain of MT plays different biological roles were studied by analyzing the difference in reactivity of the isolated domains versus different reagents. Study of the reaction between DTNB and Cd₃-beta and the reaction between Au(I)thiomalate (AuSTm) and Zn₄-alpha and Zn₃-beta confirms that the properties of the holoprotein reaction are associated with the alpha- and beta-domains. The biphasic reactions of AuSTm and DTNB can successfully be decomposed into component reactions that sum to yield the overall reaction.

• The alkylating agent N-ethylmaleimide (NEM) is the first electrophile known to react reversibly at the metal-bond thiolates of Zn7MT. The reaction was followed by monitoring the release of bound metal ions using metallochromic dyes. It goes through partially alkylated intermediates and requires a large excess of NEM to reach completion. Then, it can be reverse by addition of 2-mercaptoethanol regenerating the zinc-binding capacity of MT.

• Residues 49-61 of rabbit liver MT-II_49-61, which include the only sequential four cysteines bound to the same metal ion (Cd^I) were chemically synthesized. Its Cd-derivative was physicochemically characterized (¹¹³Cd NMR, UV-vis, CD, ES-MS, LSIMS) and its reactivity toward several organic complexes was analyzed. Molecular Mechanics calculations indicate that the folding of the polypeptide chain around the cadmium in the Cd-Pep_49-61 is the same found in MT, indicating that it does not require residues 32-49 for stabilization. Then, it exhibits properties consistent with a role in Cd-induced MT folding.

• To examine the possible influence of (1) the position of the cysteine residues and (2) the steric and electrostatic effects of neighboring amino acids on the folding and stability of MT clusters, the stable lobster beta_C and beta_N domains (each having 9 cysteines and binding 3 M²⁺) and a modified domain beta_C-N (in which the cysteines of the C-terminal domain are relocated so they are spaced as in the N-terminal domain) have been synthesized and characterized (¹¹³Cd NMR, UV-vis, CD, ES-MS, gel-filtration chromatography and chemical reactions). The synthetic native domains (Cd₃-beta_C and Cd₃-beta_N) show metal binding properties and reactivity similar to the holo-protein, but the modified Cd₃-beta_C-N domain is unusually reactive and in the presence of Chelex aggregates to a Cd₅(beta_C-N)₂ dimer. These difference in structure and reactivity demonstrate that the requirements for formation of a stable beta cluster are more stringent than simply the sequential positions of the cysteines along the peptide chain and include specific interactions with neighboring amino acids.

  
  

Doctoral Work: Electrochemical Characterization of Metallothionein

• Spectroscopical and electrochemical characterization of the metal-cysteine interactions in metallothionein and the peptide fragment 56-61 (FT) of MT-II. A diffusion controlled mechanism at the surface of the electrode was proposed which involves two different electrochemical systems (1) the oxidation of the mercury electrode in the presence of the thiolate groups contained in the polypeptides (being these groups either bound or not to metal cations depending upon the pH) and (2) the reduction of the cations at the mercury electrode. Two different Cd²⁺ or Zn²⁺ complexes were identify in MT and the FT which interconverted upon pH variations and the presence or not of excess of metal cations in solution. The apparent stability constants were also estimated from the delta E⁰ values observed.

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