20 research outputs found
ESI-MS and thermal melting studies of nanoscale platinum(ii) metallomacrocycles with DNA
The hydrophilic, long-chain diamine PEGda (O,Oā²-bis(2-aminoethyl)octadeca(ethylene glycol)), when complexed with cis-protected Pt(II) ions afforded water-soluble complexes of the type [Pt(N,N)(PEGda)](NO3)2 (N,N = N,N,Nā²,Nā²-tetramethyl-1,2-diaminoethane (tmeda), 1,2-diaminoethane (en), and 2,2ā²-bipyridine (2,2ā²-bipy)) featuring unusual 62-membered chelate rings. Equimolar mixtures containing either the 16-mer duplex DNA D2 or the single-stranded D2a and [Pt(N,N)(PEGda)]2+ were analyzed by negative-ion ESI-MS. Analysis of D2-Pt(II) mixtures showed the formation of 1:1 adducts of [Pt(en)(PEGda)]2+, [Pt(tmeda)(PEGda)]2+ and the previously-described metallomacrocycle [Pt2(2,2ā²-bipy)2{4,4ā²-bipy(CH2)44,4ā²-bipy}2]8+ with D2; the dinuclear species bound to D2 most strongly, consistent with its greater charge and aromatic surface area. D2 formed 1:2 complexes with the acyclic species [Pt(2,2ā²-bipy)(Mebipy)2]4+ and [Pt(2,2ā²-bipy)(NH3)2]2+. Analyses of D2a-Pt(II) mixtures gave results similar to those obtained with D2, although fragmentation was more pronounced, indicating that the nucleobases in D2a play more significant roles in mediating the decomposition of complexes than those in D2, in which they are paired in a complementary manner. Investigations were also conducted into the effects of selected platinum(II) complexes on the thermal denaturation of calf thymus DNA (CT-DNA) in buffered solution. Both [Pt2(2,2ā²-bipy)2{4,4ā²-bipy(CH2)64,4ā²-bipy}2]8+ and [Pt(2,2ā²-bipy)(Mebipy)2]4+ stabilized CT-DNA. In contrast, [Pt(tmeda)(PEGda)]2+ and [Pt(en)(PEGda)]2+ (as well as free PEGda) caused negligible changes in melting temperature (ĪTm), suggesting that these species interact weakly with CT-DNA
ESI-MS and thermal melting studies of nanoscale platinum(ii) metallomacrocycles with DNA
Mass spectrometric studies of non-covalent biomolecular complexes
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+ \u3e [Ru(phen)2(dpqC)]2+ \u3e [Ru(phen)2(dpq)]2+ \u3e[Ru(phen)2(pda)]2+ \u3e [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 Īµ (Īµ186, 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 Īµ186 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 Īµ186. The ability of the metal ions to enhance Īµ186 enzymatic activity was found to follow the order: Mn2+ \u3e\u3e Zn2+ \u3e 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
EX1 hydrogen-deuterium exchange in an all-helical protein and its cyclized derivative at neutral pH
The all-helical protein, DnaBN, exhibited EX1 type hydrogen exchange at pH 7.2. Approximately 45 protons were exchanged relatively rapidly, while an additional ā¼50 protons exchanged more slowly. The rates of exchange for these slowly exchanging proton
Comparison of mass spectrometry and other techniques for probing interactions between metal complexes and DNA
Electrospray ionization mass spectrometry (ESI-MS) was used to study the binding interactions of two series of ruthenium complexes, [Ru(phen)2L]2+ and [RuLā²2(dpqC)]2+, to a double stranded DNA hexadecamer, and derive orders of relative binding affinity. These were shown to be in good agreement with orders of relative binding affinity derived from absorption and circular dichroism (CD) spectroscopic examination of the same systems and from DNA melting curves. However, the extent of luminescence enhancement caused by the addition of DNA to solutions of the ruthenium complexes showed little correlation with orders of binding affinity derived from ESI-MS or any of the other techniques. Overall the results provide support for the validity of using ESI-MS to investigate non-covalent interactions between metal complexes and DNA
ESI-MS and thermal melting studies of nanoscale platinum(II) metallomacrocycles with DNA
The hydrophilic, long-chain diamine PEGda (O,Oā²-bis(2-aminoethyl) octadeca(ethylene glycol)), when complexed with cis-protected Pt(ii) ions afforded water-soluble complexes of the type [Pt(N,N)(PEGda)](NO3)2 (N,N = N,N,Nā²,Nā²-tetramethyl-1,2- diamin
EX1 hydrogen-deuterium exchange in an all-helical protein and its cyclized derivative at neutral pH
The all-helical protein, DnaBN, exhibited EX1 type hydrogen exchange at pH 7.2. Approximately 45 protons were exchanged relatively rapidly, while an additional ā¼50 protons exchanged more slowly. The rates of exchange for these slowly exchanging protons were the same, demonstrating that the slowest exchange events represent global unfolding. EX1 behavior is uncommon for native proteins. The protein was cyclized by joining the N- and C-termini through peptide linkers that were three, four, five or nine amino acids long. The corresponding ālinearā proteins were extended by addition of the same amino acids to give proteins of identical amino acid composition as their cyclized versions but differing by the mass of a water molecule. All of the proteins unfolded approximately five times faster in 10 mM compared with 100 mM ammonium acetate. In all cases, the cyclized proteins showed slower rates of amide proton exchange related to global unfolding than their linear counterparts by a factor of approximately 7- to 12-fold. Interestingly, the rate of exchange for the slowly exchanging protons decreased for both the linear and cyclized proteins as linker length increased, and this correlated with predictions that the C-terminal helix of the protein would be extended by addition of these extra amino acids. This indicates that lengthening of this helix leads to a modest increase in stability of DnaBN
Mass spectrometric studies of non-covalent binding interactions between metallointercalators and DNA
Over the past 2 decades there has been increasing interest in metal complexes that bind non-covalently to DNA, driven in part by a host of potential applications for molecules that can accomplish this task with high affinity and selectivity. As a result many workers have used a wide variety of experimental techniques, several of which are discussed in other chapters of this book, to unravel the details of the precise intermolecular interactions involved. Here we discuss one of the most recent additions to the armory of techniques used by chemists to interrogate metal complex/ DNA interactions. For the majority of its existence mass spectrometry (MS) has proven to be of enormous advantage to chemists by virtue of its ability to provide the molecular weights of compounds as well as structural information via fragmentation patterns. However, the high energies associated with many earlier MS techniques which result in fragmentation of covalent bonds, prevent its application for studying weaker intermolecular interactions. The advent of soft ionisation methods such as matrix assisted laser desorption ionisation (MALDI) and electrospray ionisation (ESI) has revolutionised mass spectrometric analysis of biomolecules, by allowing these normally fragile molecules to be introduced into the gas phase for analysis with minimal, if any, fragmentation. It was then recognised that ESIāMS, in particular, might be suitable for investigating non-covalent interactions between small molecules and either proteins or nucleic acids. This was confirmed by a number of early studies involving organic intercalators and minor groove binding ligands, prompting our interest in evaluating ESIāMS as a tool for studying non-covalent interactions between metal complexes and DNA. This chapter contains a discussion of the basic principles behind ESIāMS that enable it to introduce representative samples of solutions containing metal complexes and DNA into the gas phase for analysis. This will be followed by a discussion of the results that can be obtained using this method, drawing heavily on studies performed in our laboratory
Proteomic dissection of DNA polymerization
DNA polymerases replicate the genome by associating with a range of other proteins that enable rapid, high-fidelity copying of DNA. This complex of proteins and nucleic acids is called the replisome. Proteins of the replisome must interact with other networks of proteins, such as those involved in DNA repair. Many of the proteins involved in DNA polymerisation and the accessory proteins are known, but the array of proteins they interact with, and the spatial and temporal arrangement of these interactions is a current research topic. Mass spectrometry is a technique that can be used to identify the sites of these interactions and to determine the precise stoichiometries of binding partners in a functional complex. A complete understanding of the macromolecular interactions involved in DNA replication and repair may lead to discovery of new targets for antibiotics against bacteria and biomarkers for diagnosis of diseases such as cancer in humans
Probing DNA selectivity of ruthenium metallointercalators using ESI mass spectrometry
ESI mass spectra show that up to five ruthenium molecules can bind non-covalently to double stranded 16mer DNA, and provide information on the relative affinity and DNA sequence selectivity of different ruthenium complexes