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Lobster Metallothionein 113Cd3-b Domain Solvent Atom Exposure Patterns Studied by NMR Spectroscopy:
Impact on Metal-Thiolate Cluster Reactivity

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Sulfur Coverage and metal-thiolate reactivity

Studies of the reactivity of isolated lobster Cd3b domains with DTNB revealed biphasic reactions for both clusters (Figure 8). With Cd3bN, 6 of 9 thiolate groups react faster; with Cd3bC, only 3 react in the faster step, indicating different reactivity for distinct sulfur atoms within one domain [6]. bar.gif
Figure 8:Reaction of 4 mM Cd3 bC (red ) and Cd3bN ( green ) with 0.5 mM DTNB at pH 7.5 in 5 mM Tris and 0.1M KCl. Displayed is the absorbance at 412nm corresponding to the TNB formed in the reaction. Figure modified from [6].
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A close inspection of the NMR structure with respect to exposure of sulphur atoms reveals that the bN domain exhibits a much more exposed metal-sulphur cluster when compared with theb C (Figure 8) corresponding to surface exposed surfaces of 45.5 and 17.0 A2, respectively (Figure 9). On the basis of the analysis of surface exposure of sulfur atoms, one of the Cd ions and 3 of its sulfhydryl ligands of Cd3bN are effectively buried, consistent with their slower reactivity. In Cd3bC, two Cd ions and their thiolate ligands are buried and are thought to be less reactive with DTNB.
Another interesting observation emerges when the contributions of backbone versus sidechains to the total coverage of the sulphur atoms are considered. In the case of bN most of the contributions arise from Cys residues and the backbone alone, only the side chains of two residues (S24 and R26) further reduce the exposure of thiolates. In the case of bC the influence of side chains is somewhat larger with a total of 7 residues exhibiting significant contributions (E31, K32, K42, T48, K51, P57 and T58), but the bulk of the coverage is still achieved by Cys and backbone alone (Figure 9). bar.gif
Figure 9 : Space filling drawing of the structures of the bN and b C domains. Cys residues and backbone atoms are colored by atom type. Side chains contributing to sulphur coverage are colored in orange, whereas R groups without any contribution are shown in pale yellow.
This figure was prepared using the program MOLMOL2k.1 by Koradi et al. [4]
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