Studies of viomycin, an anti-tuberculosis antibiotic: Copper(II) coordination, DNA degradation and the impact on delta ribozyme cleavage activity

Viomycin is a basic peptide antibiotic, which is among the most effective agents against multidrug-resistant tuberculosis. In this paper we provide the characteristics of its acid base properties, coordination preferences towards the Cu(II) ions, as well as the reactivity of the resulting complexes against plasmid DNA and HDV ribozyme. Careful coordination studies throughout the wide pH range allow for the characterisation of all the Cu(II)-viomycin complex species. The assignment of proton chemical shifts was achieved by NMR experiments, while the DTF level of theory was applied to support molecular structures of the studied complexes. The experiments with the plasmid DNA reveal that at the physiological levels of hydrogen peroxide the Cu(II)-viomycin complex is more aggressive against DNA than uncomplexed metal ions. Moreover, the degradation of DNA by viomycin can be carried out without the presence of transition metal ions. In the studies of antigenomic delta ribozyme catalytic activity, viomycin and its complex are shown to modulate the ribozyme functioning. The molecular modelling approach allows the indication of two different locations of viomycin binding sites to the ribozyme

Double-selective excitation techniques as a means of delineating dynamics of protein-bound small molecules

A method for delineation of dynamics of protein-bound ligands is presented that relies on measuring 'H NMR spin-lattice relaxation rates upon double-selective excitation of dipolarly connected ligand resonances. Dipolar interaction energies of proton pairs are obtained in the free and bound states. For protons at fixed distances such dipolar energies are interpreted in terms of motional correlation times of relaxation vectors. As an example the reorientational correlation time of the indole moiety was evaluated at 23.5 ps for L-tryptophan in water solution and at 27.0 ns for L-tryptophan bound to human serum albumin

NMR investigation of cell cultures: early detection of infection by Herpes simplex virus type 2 and transformation

The water proton Nuclear Magnetic Resonance (NMR) spin-lattice relaxation rates were measured in suspension of uninfected and Herpes simplex virus type 2-infected WI-38 diploid cells and shown to allow reliable detection of (i) the early signs of productive infection, and (ii) the tendency to undergo either spontaneous or virus-induced transformation. In addition, NMR investigation of several transformed cell lines currently and previously established from WI-38 cells clearly indicated that the NMR relaxation parameter is a suitable tool for assessment of the level of malignancy, as revealed by in vivo oncogenicity tests in immunosuppressed mice

1H-NMR studies on the self-association of chloroquine in aqueous solution

The concentration dependences of 1H-NMR chemical shifts and spin-lattice relaxation rates were measured for chloroquine in aqueous solution. The weak self-association constant was evaluated according to a dimerization equilibrium with the formation of self-stacked adducts (Kd = 4.52 +/- 0.68 l mol-1). The motional correlation times were evaluated for the monomer and the dimer by measuring intramolecular dipolar cross-relaxation rates of aromatic vicinal protons (tau cm = 0.06 ns and tau cd = 0.26 ns). The geometry of the stacked dimer was elucidated by measuring intermolecular dipolar cross-relaxation rates and interpreted in terms of partial superposition of quinoline moieties

The role of His-50 of α-Synuclein in binding Cu(II): pH dependence, speciation, thermodynamics and structures

Copper interaction with alpha synuclein (αS) has been shown to accelerate aggregation and oligomerization of the protein. Three different αS copper binding domain have been proposed: (i) 10 the N-terminal residues (1-9) that represent the minimal copper binding domain; (ii) the His-50 imidazole and (iii) the Asp and Glu residues within the acidic C-terminal domain. The copper coordination at the N-terminus has been extensively characterized and it is generally accepted that it provides the highest affinity site. The same does not hold for the role played by His-50 in copper binding. In this work Cu(II) coordination to peptide fragments encompassing residues 45-55 of αS 15 has been exhaustively characterized, including systems containing the inherited mutations E46K and A53T, as model peptides of the His-50 site. Through potentiometic titrations all the speciation profiles have been determined and the stability constants have been used to estimate the dissociation constants of complexes corresponding to the binding modes at pH 6.5 and 7.5. Spectroscopic analyses allowed determination of (i) the copper coordination sphere, (ii) its 20 geometry and (iii) the constraints wherefrom the 3D structural models of the copper complexes could be obtained

Interaction of Angiotensin II with the C-terminal 300-320 fragment of the rat Angiotensin II Receptor AT1a monitored by NMR.

Interaction between angiotensin II (Ang II) and the fragment peptide 300–320 (fCT300–320) of the rat angiotensin II receptor AT1a was demonstrated by relaxation measurements, NOE effects, chemical shift variations, and CD measurements. The correlation times modulating dipolar interactions for the bound and free forms of Ang II were estimated by the ratio of the nonselective and single-selective longitudinal relaxation rates. The intermolecular NOEs observed in NOESY spectra between HN protons of 9LysfCT and 6Hisang, 10PhefCT and 8Pheang, HN proton of 3TyrfCT and Hα of 4Tyrang, 5PhefCTHδ and Hα of 4Tyrang indicated that Ang II aromatic residues are directly involved in the interaction, as also verified by relaxation data. Some fCT300–320 backbone features were inferred by the CSI method and CD experiments revealing that the presence of Ang II enhances the existential probability of helical conformations in the fCT fragment. Restrained molecular dynamics using the simulated annealing protocol was performed with intermolecular NOEs as constraints, imposing an α-helix backbone structure to fCT300–320 fragment. In the built model, one strongly preferred interaction was found that allows intermolecular stacking between aromatic rings and forces the peptide to wrap around the 6Leu side chain of the receptor fragment