Structure, dynamics and hydration in drug-DNA recognition

The role of deoxyribonucleic acids in the cell has made DNA an attractive target for drug molecules. The anthracycline antitumour antibiotics are potent cytotoxic agents that have found widespread use in cancer chemotherapy. Nogalamycin binds DNA through intercalation, preferentially to 5'-TpG and 5'-CpG sites, by threading through the DNA helix and interacting with both the major and minor grooves simultaneously. In this thesis, the interaction of nogalamycin with the 5'-TpG site has been investigated using synthetic oligonucleotide duplexes and a combination of high-resolution NMR techniques and NOE-restrained molecular dynamics simulations. The solution structure of the 1: 1 complex with d(ATGCAT)2 is described with NOE data unambiguously identifying the position and orientation of the bound drug molecule, allowing conclusions to be drawn regarding the specificity for the TpG site. Binding at one TpG site sterically blocks the interaction at the symmetrically equivalent CpA site. The structural studies are extended to investigate by NMR the role of solvation in drug- DNA recognition and binding. Based on the sign and magnitude of solute-solvent NOEs, it is shown that only a small subset of water molecules visible in the crystal and MD structures are found to be bound in the solution complex, and that a number of these are involved in mediating drug-DNA interactions. The role of the dynamic network of water molecules in stabilising the complex in solution is discussed. Finally, the binding of nogalamycin at a TpG site carrying a DNA strand break has been investigated using a novel designed single-stranded intermolecular duplex consisting of two hairpins stabilised by GAA loops [d(ACGAAGTGCGAAGC)]. Although stacking of the two hairpins is weak, nogalamycin is shown to bind and stabilise a 1: 1 complex by binding at the intercalation site. The complex is discussed in terms of the mechanism by which nogalamycin is able to bind to premelted duplex DNA

Structure, dynamics and hydration in drug-DNA recognition

The role of deoxyribonucleic acids in the cell has made DNA an attractive target for drug molecules. The anthracycline antitumour antibiotics are potent cytotoxic agents that have found widespread use in cancer chemotherapy. Nogalamycin binds DNA through intercalation, preferentially to 5'-TpG and 5'-CpG sites, by threading through the DNA helix and interacting with both the major and minor grooves simultaneously. In this thesis, the interaction of nogalamycin with the 5'-TpG site has been investigated using synthetic oligonucleotide duplexes and a combination of high-resolution NMR techniques and NOE-restrained molecular dynamics simulations. The solution structure of the 1: 1 complex with d(ATGCAT)2 is described with NOE data unambiguously identifying the position and orientation of the bound drug molecule, allowing conclusions to be drawn regarding the specificity for the TpG site. Binding at one TpG site sterically blocks the interaction at the symmetrically equivalent CpA site. The structural studies are extended to investigate by NMR the role of solvation in drug- DNA recognition and binding. Based on the sign and magnitude of solute-solvent NOEs, it is shown that only a small subset of water molecules visible in the crystal and MD structures are found to be bound in the solution complex, and that a number of these are involved in mediating drug-DNA interactions. The role of the dynamic network of water molecules in stabilising the complex in solution is discussed. Finally, the binding of nogalamycin at a TpG site carrying a DNA strand break has been investigated using a novel designed single-stranded intermolecular duplex consisting of two hairpins stabilised by GAA loops [d(ACGAAGTGCGAAGC)]. Although stacking of the two hairpins is weak, nogalamycin is shown to bind and stabilise a 1: 1 complex by binding at the intercalation site. The complex is discussed in terms of the mechanism by which nogalamycin is able to bind to premelted duplex DNA

Developing Mn-doped lead sulfide quantum dots for MRI labels

Magnetic interactions of Mn2+ions in lead sulfide (PbS) nanocrystals with protons in water are probed by NMR and MRI. A thin layer of capping molecules enables free solvent diffusion to the nanocrystal surface resulting in a decrease of proton relaxation times. Magnetic resonance imaging of neuronal cell pellets exposed to (PbMn)S at non-toxic concentrations demonstrates their prospects as MRI-labels

SilE is an intrinsically disordered periplasmic ‘molecular sponge' involved in bacterial silver resistance

Ag+ resistance was initially found on the Salmonella enetrica serovar Typhimurium multi-resistance plasmid pMG101 from burns patients in 1975. The putative model of Ag+ resistance, encoded by the sil operon from pMG101, involves export of Ag+ via an ATPase (SilP), an effluxer complex (SilCFBA) and a periplasmic chaperon of Ag+ (SilE). SilE is predicted to be intrinsically disordered. We tested this hypothesis using structural and biophysical studies and show that SilE is an intrinsically disordered protein in its free apo-form but folds to a compact structure upon optimal binding to six Ag+ ions in its holo-form. Sequence analyses and site-directed mutagenesis established the importance of histidine and methionine containing motifs for Ag+-binding, and identified a nucleation core that initiates Ag+-mediated folding of SilE. We conclude that SilE is a molecular sponge for absorbing metal ions