Year

2006

Degree Name

Doctor of Philosophy (PhD)

Department

Department of Chemistry - Faculty of Science

Abstract

Electrospray ionisation mass spectrometry (ESI-MS) was employed to investigate non-covalent associations of macromolecules with ligands, metal ions and other macromolecules. Firstly, ESI-MS was used to examine the interactions of six ruthenium compounds with three different DNA sequences (D1, D2 and D3). The relative binding affinities of these ruthenium compounds towards dsDNA was determined to be: [Ru(phen)2(dppz)]2+ . [Ru(phen)2(dpqMe2)]2+ > [Ru(phen)2(dpqC)]2+ > [Ru(phen)2(dpq)]2+ >[Ru(phen)2(pda)]2+ > [Ru(phen)3]2+. This order was in good agreement with that obtained from DNA melting temperature experiments. Competition experiments involving ruthenium compounds and organic drugs were also conducted to obtain information about the DNA binding modes of the ruthenium compounds. These studies provide strong support for the routine application of ESI-MS as a tool for analysis of non-covalent complexes between metallointercalators and dsDNA. ESI-MS also proved to be a rapid and efficient tool for investigation of interactions between the N-terminal domain of ε (ε186, the exonuclease proofreading subunit of E. coli DNA) and three different metal ions (Mn2+, Zn2+ and Dy3+). The dissociation constants (Kd) for binding of Mn2+, Zn2+ and Dy3+ to ε186 were determined from ESI-MS data to be 38.5 x 10-6, 3.7 x 10-6 and 2.0 x 10-6 M, respectively. Despite binding the least tightly to the protein, incorporation of Mn2+ into the enzyme resulted in the highest enzymatic activity as measured by spectrophotometric studies. This suggested that Mn2+ is possibly the native metal ion present in ε186. The ability of the metal ions to enhance ε186 enzymatic activity was found to follow the order: Mn2+ >> Zn2+ > Dy3+. The results of these experiments also provided evidence that the presence of two divalent metal ions was essential for efficient enzyme-catalysed hydrolysis. The distribution of different oligomeric forms of wild-type E. coli DnaB helicase and DnaB helicase mutants (F102E, F102H, F102W and D82N) was examined using a factory-modified Q-ToF mass spectrometer equipped with a 32,000 m/z quadrupole. Previous experiments showed that the heptameric form of the wild-type protein was favoured in the presence of methanol (30% v/v). In the current work, mixtures of hexamer, heptamer, decamer and dodecamer were observed in solutions containing 1000 mM NH4OAc, 1 mM Mg2+ and 0.1 mM ATP, pH 7.6. When the proteins were prepared in solutions containing a lower concentration of Mg2+ (0.1 mM), only the hexameric form was observed for all proteins except D82N, which showed a mixture of hexamer and heptamer. These observations suggest that the higher order structures were stabilised at high concentrations of Mg2+. In addition, the hexamers of DnaB and mutants ((DnaB)6, (F102W)6 and (D82N )6) formed complexes with four to six molecules of the helicase loading partner, DnaC. ESI-MS was used in conjunction with hydrogen/deuterium exchange studies to probe the unfolding mechanisms of linear and cyclised DnaB-N (the N-terminal domain of DnaB helicase) containing linkers comprised of different numbers of amino acid residues (3, 4, 5 and 9). The unfolding rates for all the cyclised proteins were about ten-fold slower than for the corresponding linear proteins. These observations suggest that enhancement of protein stability against unfolding could be achieved through cyclisation. Furthermore, the HDX data showed that all the proteins examined exhibited a rare EX1 mechanism at near neutral pH.

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