1H NMR Study of the Enantioselective Binding of λ- and Δ-[Ru(bpy)2(m-bpy-GHK)]Cl2 to the Deoxynucleotide Duplex d(5'-C1G2C3G4A5A6T7T8C9G10C11G12-3')2

The interaction of the diastereomeric complexes Λ- and Δ - [Ru(bpy)2(m - GHK)]Cl2, (GHK = glycine-histidine-lysine) to the deoxynucleotide duplex d(5'-CGCGAATTCGCG-3')2 was studied by means of 1H NMR spectroscopy. The diastereomers interact with the oligonucleotide duplex differently. The Δ - [Ru(bpy)2 (m - GHK)]Cl2 is characterized by major groove binding close to the central part of the oligonucleotide, with both the peptide and the bipyridine ligand of the complex involved in the binding. The λ - [Ru(bpy)2 (m - bpy - GHK)]C2 binds loosely, approaching the helix from the minor groove. The NMR analysis shows that the peptide (GHK) binding has a determinative role in the interactions of both diastereomers with the oligonucleotide

Copper binding to the N-terminal metal-binding sites or the CPC motif is not essential for copper-induced trafficking of the human Wilson protein (ATP7B)

The Wilson protein (ATP7B) is a copper-translocating P-type ATPase that mediates the excretion of excess copper from hep-atocytes into bile. Excess copper causes the protein to traffic from the TGN (trans-Golgi network) to subapical vesicles. Using site-directed mutagenesis, mutations known or predicted to abrogate catalytic activity (copper translocation) were introduced into ATP7B and the effect of these mutations on the intracellular traf-ficking of the protein was investigated. Mutation of the critical aspartic acid residue in the phosphorylation domain (DKTGTIT) blocked copper-induced redistribution of ATP7B from the TGN, whereas mutation of the phosphatase domain [TGE (Thr-Gly-Glu)] trapped ATP7B at cytosolic vesicular compartments. Our findings demonstrate that ATP7B trafficking is regulated with its copper-translocation cycle, with cytosolic vesicular localization associated with the acyl-phosphate intermediate. In addition, mut-ation of the six N-terminal metal-binding sites and/or the trans-membrane CPC (Cys-Pro-Cys) motif did not suppress the consti-tutive vesicular localization of the ATP7B phosphatase domain mutant. These results suggested that copper co-ordination by these sites is not essential for trafficking. Importantly, copper-chelation studies with these mutants clearly demonstrated a requirement for copper in ATP7B trafficking, suggesting the presence of an additional copper-binding site(s) within the protein. The results presented in this report significantly advance our understanding of the regulatory mechanism that links copper-translocation activity with copper-induced intracellular trafficking of ATP7B, which is central to hepatic and hence systemic copper homoeostasis

Environmental Influence on Zn–Histidine Complexes under No-Packing Conditions

This paper describes a combined structural analysis of the Zn-histidine complex, using two different and complementary techniques, X-ray absorption spectroscopy (XAS) and surface X-ray diffraction, paying special attention to the environmental conditions. The current procedure for investigating macromolecules consists of examining simple molecules that exhibit properties similar to those of the larger ones, whose functionality is totally related to the atomic structure. The detailed study of the bonding structure formed by zinc and histidine amino acids is motivated by the fact that this material serves as a model for metalloproteins, such as in metalloproteinase, acting as active sites in enzymatic or structural functions. For XAS modeling, Zn-histidine complexes were dissolved in several aqueous solutions, over a wide pH range. Correlations among the degree of protonation, the steric impediment, and the multiple combinations of the histidine amino acid have been found. For the diffraction study, high-quality crystals grown by the seeding method in a supersaturated solution have been studied, and the samples for the surface diffraction study were mounted on a cell specially designed for solid-liquid or solid-gas interface analysis. The surface structural model was built from XAS results. In both cases, the obtained structures are compared with the bulk one, showing atomic differences and highlighting the importance of the environment in which the complex is studied. © 2014 American Chemical Society.The financial support of the Consejo Superior de Investigaciones Cientifícas and Spanish Ministerio de Ciencia e Innovación (PI201060E013 and MAT2011-23758) is also acknowledged.Peer Reviewe