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• Articles by J.G. Duguid
• Articles by V.A. Bloomfield

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• Raman spectroscopy of DNA-metal complexes. II. The thermal d...

  Raman spectroscopy of DNA-metal complexes. II. The thermal denaturation of DNA in the presence of Sr2+, Ba2+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, and Cd2+ Biophysical Journal, Volume 69, Issue 6, 1 December 1995, Pages 2623-2641 J.G. Duguid, V.A. Bloomfield, J.M. Benevides and G.J. Thomas Abstract Differential scanning calorimetry, laser Raman spectroscopy, optical densitometry, and pH potentiometry have been used to investigate DNA melting profiles in the presence of the chloride salts of Ba2+, Sr2+, Mg2+, Ca2+, Mn2+, Co2+, Ni2+, and Cd2+. Metal-DNA interactions have been observed for the molar ratio [M2+]/[PO2-] = 0.6 in aqueous solutions containing 5% by weight of 160 bp mononucleosomal calf thymus DNA. All of the alkaline earth metals, plus Mn2+, elevate the melting temperature of DNA (Tm > 75.5 degrees C), whereas the transition metals Co2+, Ni2+, and Cd2+ lower Tm. Calorimetric (delta Hcal) and van't Hoff (delta HVH) enthalpies of melting range from 6.2–8.7 kcal/mol bp and 75.6–188.6 kcal/mol cooperative unit, respectively, and entropies from 17.5 to 24.7 cal/K mol bp. The average number of base pairs in a cooperative melting unit (<nmelt>) varied from 11.3 to 28.1. No dichotomy was observed between alkaline earth and transition DNA-metal complexes for any of the thermodynamic parameters other than their effects on Tm. These results complement Raman difference spectra, which reveal decreases in backbone order, base unstacking, distortion of glycosyl torsion angles, and rupture of hydrogen bonds, which occur after thermal denaturation. Raman difference spectroscopy shows that transition metals interact with the N7 atom of guanine in duplex DNA. A broader range of interaction sites with single-stranded DNA includes ionic phosphates, the N1 and N7 atoms of purines, and the N3 atom of pyrimidines. For alkaline earth metals, very little interaction was observed with duplex DNA, whereas spectra of single-stranded complexes are very similar to those of melted DNA without metal. However, difference spectra reveal some metal-specific perturbations at 1092 cm-1 (nPO2-), 1258 cm-1 (dC, dA), and 1668 cm-1 (nC==O, dNH2 dT, dG, dC). Increased spectral intensity could also be observed near 1335 cm-1 (dA, dG) for CaDNA. Optical densitometry, employed to detect DNA aggregation, reveals increased turbidity during the melting transition for all divalent DNA-metal complexes, except SrDNA and BaDNA. Turbidity was not observed for DNA in the absence of metal. A correlation was made between DNA melting, aggregation, and the ratio of Raman intensities I1335/I1374. At room temperature, DNA-metal interactions result in a pH drop of 1.2–2.2 units for alkaline earths and more than 2.5 units for transition metals. Sr2+, Ba2+, and Mg2+ cause protonated sites on the DNA to become thermally labile. These results lead to a model that describes DNA aggregation and denaturation during heating in the presence of divalent metal cations; 1) The cations initially interact with the DNA at phosphate and/or base sites, resulting in proton displacement. 2) A combination of metal-base interactions and heating disrupts the base pairing within the DNA duplex. This allows divalent metals and protons to bind to additional sites on the DNA bases during the aggregation/melting process. 3) Strands whose bases have swung open upon disruption are linked to neighboring strands by metal ion bridges. 4) Near the midpoint of the melting transition, thermal energy breaks up the aggregate. We have no evidence to indicate whether metal ion cross-bridges or direct base-base interactions rupture first. 5) Finally, all cross-links break, resulting in single-stranded DNA complexed with metal ions. Abstract | PDF (1729 kb)

• Fourier Transform Infrared Study of the Effect of Different ...

  Fourier Transform Infrared Study of the Effect of Different Cations on Bacteriorhodopsin Protein Thermal Stability Biophysical Journal, Volume 82, Issue 3, 1 March 2002, Pages 1598-1606 Colin D. Heyes, Jianping Wang, Laurie S. Sanii and Mostafa A. El-Sayed Abstract The effect of divalent ion binding to deionized bacteriorhodopsin (dI-bR) on the thermal transitions of the protein secondary structure have been studied by using temperature-dependent Fourier transform infrared (FT-IR) spectroscopy. The native metal ions in bR, Ca, and Mg, which we studied previously, are compared with Mn, Hg, and a large, synthesized divalent organic cation, ((Et)N)Bu. It was found that in all cases of ion regeneration, there is a pre-melting, reversible conformational transition in which the amide frequency shifts from 1665 to 1652cm. This always occurs at ∼80°C, independent of which cation is used for the regeneration. The irreversible thermal transition (melting), monitored by the appearance of the band at 1623cm, is found to occur at a lower temperature than that for the native bR but higher than that for acid blue bR in all cases. However, the temperature for this transition is dependent on the identity of the cation. Furthermore, it is shown that the mechanism of melting of the organic cation regenerated bR is different than for the metal cations, suggesting a difference in the type of binding to the protein (either to different sites or different binding to the same site). These results are used to propose specific direct binding mechanisms of the ions to the protein of deionized bR. Abstract | Full Text | PDF (164 kb)

• Application of Polyelectrolyte Theories for Analysis of DNA ...

  Application of Polyelectrolyte Theories for Analysis of DNA Melting in the Presence of Na and Mg Ions Biophysical Journal, Volume 75, Issue 6, 1 December 1998, Pages 3041-3056 Nikolay Korolev, Alexander P. Lyubartsev and Lars Nordenskiöld Abstract Numerical calculations, using Poisson-Boltzmann (PB) and counterion condensation (CC) polyelectrolyte theories, of the electrostatic free energy difference, Δ, between single-stranded (coil) and double-helical DNA have been performed for solutions of NaDNA+NaCl with and without added MgCl. Calculations have been made for conditions relevant to systems where experimental values of helix coil transition temperature () and other thermodynamic quantities have been measured. Comparison with experimental data has been possible by invoking values of for solutions containing NaCl salt only. Resulting theoretical values of enthalpy, entropy, and heat capacity (for NaCl salt-containing solutions) and of as a function of NaCl concentration in NaCl+MgCl solutions have thus been obtained. Qualitative and, to a large extent, quantitative reproduction of the experimental , Δ, Δ, and Δ values have been found from the results of polyelectrolyte theories. However, the quantitative resemblance of experimental data is considerably better for PB theory as compared to the CC model. Furthermore, some rather implausible qualitative conclusions are obtained within the CC results for DNA melting in NaCl+MgCl solutions. Our results argue in favor of the Poisson-Boltzmann theory, as compared to the counterion condensation theory. Abstract | Full Text | PDF (227 kb)

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Abstract

In an accompanying paper we reported the use of differential scanning calorimetry and optical densitometry to characterize the melting and aggregation of 160 bp fragments of calf thymus DNA during heating in the presence of divalent metal cations. Aggregation is observed as thermal denaturation begins and becomes more extensive with increasing temperature until the melting temperature Tm is reached, after which the aggregates dissolve extensively. The order of effectiveness of the metals in inducing aggregation is generally consistent with their ability to induce melting: Cd > Ni > Co > Mn approximately Ca > Mg. Under our experimental conditions (50 mg/ml DNA, 100 mM MCl2, [metal]/[DNA phosphate] approximately 0.6), no measurable aggregates were observed for BaDNA or SrDNA. In this paper we show that the Shibata-Schurr theory of aggregation in the thermal denaturation region provides a good model for our observations. Free energies of cross-linking, induced by the divalent cations, are estimated to be between 34% and 38% of the free energies of base stacking. The ability of a divalent metal cation to induce DNA aggregation can be attributed to its ability to disrupt DNA base pairing and simultaneously to link two different DNA sites.