Determination of the solution three dimensional structure of chemically-synthesized native 113Cd3S9 domain of Lobster Metallothionein by NMR Spectroscopy
Amalia Muņoz*, F. Holger Forsterling, C. Frank Shaw III+ and David H. Petering
Solid Phase methods using Fmoc-protected amino acids were used to synthesize the following b-domain sequences:
After the cleavage, the synthesized domains were purified by reverse phase-HPLC. Sequential treatment with Hg(CH3COO)2 and beta-mer-captoethanol removes the Acm-groups of the cysteine residues and results in the corresponding apo-beta domains. ES-MS carried out at each stage of peptide deblocking of these synthetic sequences and amino acid analysis of the resulting apo-peptides show a good agreement between the experimental and theoretical values.
The cadmium / cysteine ratio calculated from the DTNB reaction and AAS analysis resulted in ~3.0, as expected for the holoprotein. UV-spectra for the Cd3betaC synthetic native domains showed an absorption band at ~250 nm characteristic of metal to ligand (Cd-S) charge transfers. And, the CD-spectrum with a band center at 260 nm indicates that clusters like those of observed for the holoprotein are formed. The pH-titration curve and the pH of half dissociation for Cadmium are in very good agreement with those observed in the case of the holoprotein.
The 113Cd-1D NMR chemical shifts for the 113Cd3betaC domain indicates that the Cd2+ is tetracoordinated to four cysteines with some cysteines bridging between two Cd2+. The cadmium chemical shifts exhibit by the synthetic native lobster domain, 113Cd3betaC are in good agreement with the values corresponding to the betaC-domain of the holoprotein (lobster Cd6-MT) previously reported. Thus, 113Cd-NMR and 1H NMR(which shows a good dispersion at the amide region) confirm that the synthetic native domain has formed stable clusters similar to those found for the natural holoprotein.
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