Molecular mechanics and dynamics calculations on (dA)10.(dT)10 incorporating distance constraints derived from NMR relaxation measurements

Structural constraints derived from proton NMR relaxation measurements on poly(dA).poly(dT) in the form of interproton separations and orientation have been combined with molecular mechanics and annealed molecular dynamics calculations to derive a model for the solution-state structure of this molecule. Three different possible starting configurations, including the standard A and B forms of Arnott and Hukins [Arnott, S., & Hukins, D. W. L. (1972) Biochem. Biophys. Res. Commun. 47, 1506-1509] and the heteronomous (H) structure [Arnott, S., Chandrasekaran, R., Hall, I. H., & Puigjaner, L. C. (1983) Nucleic Acids Res. 11, 4141-4155], were examined. Both the B- and H-DNA structures converged to the same B-like structure (approximately C2'-endo conformation on both the A and T sugars, glycosidic bond torsional angle of 63-73 degrees) with the same energies and average helical parameters that gave good fits of the NMR relaxation rates. This model also accounts for the experimental observation [Behling, R. W., & Kearns, D. R. (1986) Biochemistry 25, 3335-3346] that the AH2 proton interacts more strongly with the H1' sugar proton on the T strand than on the A strand. Although the helix repeat angle (39 degrees) is larger than that for standard B-DNA (36 degrees), this does not result in a significantly smaller minor groove, as monitored by the interstrand P-P separation. Calculations starting with the A-DNA structure lead to a very high energy structure that gave a poorer fit of the NMR data.     

Oxygen radical-mediated DNA damage by redox-active Cr(III) complexes.

The mechanism of DNA damage induced by Cr(III) complexes is currently unknown even though it is considered to be the ultimate biologically active oxidation state of chromium. In this study, we have employed the Salmonella reversion assay to identify mutagenic Cr(III) complexes. Cyclic voltammetry was used to differentiate the redox kinetics between mutagenic and selected nonmutagenic Cr(III) species. Plasmid relaxation of supercoiled DNA was employed to show in vitro interactions with plasmid DNA and correlate the interactions with the electrochemical behavior and biological activity. The results of this study demonstrate that the mutagenic Cr(III) complexes identified in the Salmonella reversion assay display characteristics of reversibility and positive shifts of the Cr(III)/Cr(II) redox couple consistent with the ability of these Cr(III) complexes to serve as cyclical electron donors in a Fenton-like reaction. These same mutagenic complexes display an ability to relax supercoiled DNA in vitro, presumably by the induction of single-strand breaks. Nonmutagenic complexes were selected to test different ligands to determine how the ligand directs the activity of Cr(III) complexes. All nonmutagenic complexes tested thus far have shown classical irreversibility, more negative reduction potentials, and an inability to relax supercoiled plasmid DNA. These results suggest that the mechanism by which chromium complexes potentiate mutagenesis involves an oxygen radical as an active intermediate. These data also demonstrate the effect of associated ligands with regard to the ability of a metal to generate an active redox center.     

NMR studies of primary and secondary sites of parvalbumins using the two paramagnetic probes Gd (III) and Mn (II)

The binding of cations by parvalbumins was studied by the proton relaxation enhancement (PRE) method using the paramagnetic probes Gd(III) and Mn(II). Gd(III) appears as a specific probe of the primary sites CD and EF with the following binding parameters: n = 2, KdGd = 0.5 x 10(-11) M and epsilon b = 2.3. The low value of epsilon b is the result of a nearly complete dehydration of the protein bound ions. Competition experiments between Gd(III) and various diamagnetic cations show the following order of affinity for the EF and CD sites: Mg2+ less than Zn2+ less than Sr2+ less than Ca2+ less than Cd2+ less than La3+ less than or equal to Gd3+. Mn 2+ is a specific probe of a secondary site with the following binding parameters: n = 1, KdMn = 0.6 x 10(-3) M and epsilon b = 17. The high value of epsilon b suggests that the protein bound Mn(II) has retained most of its hydration shell. Competition experiments between (Mn(II) and different cations show similar affinities for this site: Ca2+ less than or equal to Mg2+ less than or equal to Cd2+ less than or equal to Mn2+. This secondary site is located near the EF primary site.     

The preparation and characterization of Cr(III) and Co(III) complexes of GDP and GTP and their interactions with avian phosphoenolpyruvate carboxykinase

The exchange inert coordination complexes, Cr(H2O)4GDP, Cr(H2O)4GTP, Cr(NH3)4GDP, Cr(NH3)4GTP, Co(NH3)4GDP, and Co(NH3)4GTP have been synthesized and characterized. The lambda and delta coordination isomers of Cr(H2O)4GDP, Cr(NH3)4GDP, and the four Cr(H2O)4GTP isomers have been separated by reverse phase HPLC and characterized by their CD spectra. While the isomers of Co(NH3)4GTP have not been successfully separated, 31P NMR spectroscopy reveals the presence of the lambda and delta forms. The complexes, Cr(H2O)4GDP, Co(NH3)4GDP, Cr(H2O)4GTP, and Co(NH3)4GTP, are linear competitive inhibitors of avian phosphoenolpyruvate carboxykinase. The Ki values of 30 microM, 540 microM, 40 microM, and 12 microM, respectively, were determined for these complexes using Mn-IDP as the nucleotide substrate in the phosphoenolpyruvate carboxylation direction or Mn-ITP as nucleotide substrate for the oxalacetate decarboxylation reaction. The lambda and delta isomers of Cr(H2O)4 GDP show little specificity (a twofold maximum difference in Ki) for the enzyme. The isomeric forms of Cr(H2O)4 GTP demonstrate no observed stereoselectivity of interaction with the enzyme. All of the complexes tested, except for Cr(NH3)4GDP and Co(NH3)4GDP, which have larger Ki values, are good substrate analogs for P-enolpyruvate carboxykinase. When the substrate is Mn-GTP, fixed at 0.2 mM at pH 6.0, enzyme activity is stimulated two- to two and a half-fold by Cr(H2O)4GTP. A Dixon plot reveals that the stimulatory effect is saturated at 0.4 mM Cr(H2O)4GTP. The interaction of the enzyme with Cr(H2O)4GTP appears to produce a "memory" effect which is manifest with guanosine nucleotide substrates, but which is not observed with the alternative substrate Mn-ITP.     

Interaction of La (III) and Tb (III) ions with purine nucleotides: evidence for metal chelation (N-7-M-PO3) and the effect of macrochelate formation on the nucleotide sugar conformation.

The interaction of the La (III) and Tb (III) ions with adenosine-5'-monophosphate (5'-AMP), guanosine-5'-monophosphate (5'-GMP), and 2'-deoxyguanosine-5'-monophosphate (5'-dGMP) anions with metal/nucleotide ratios of 1 and 2 has been studied in aqueous solution in acidic and neutral pHs. The solid complexes were isolated and characterized by Fourier transform ir and 1H-nmr spectroscopy. The lanthanide (III)-nucleotide complexes are polymeric in nature both in the solid and aqueous solutions. In the metal-nucleotide complexes isolated from acidic solution, the nucleotide binding is via the phosphate group (inner sphere) and an indirect metal-N-7 interaction (outer-sphere) with the adenine N-1 site protonated. In the complexes obtained from neutral solution, metal chelation through the N-7 and the PO3(2-) group is prevailing. In aqueous solution, an equilibrium between the inner and outer sphere metal-nucleotide interaction has been observed. The ribose moiety shows C2'-endo/anti pucker in the free AMP anion and in the lanthanide (III)-AMP complexes, whereas the GMP anion with C2'-endo/anti sugar conformation exhibits a mixture of the C2'-endo/anti and C3'-endo/anti sugar puckers in the lanthanide (III)-GMP salts. The deoxyribose has O4'-endo/anti sugar pucker in the free dGMP anion and a C3'-endo/anti, in the lanthanide (III)-dGMP complexes.     

Differential effects of Co(III), Ni(II), and Ru(III) amine complexes on Sindbis virus

Transition metal complexes [Co(cyclen)(NH₃)₂](ClO₄)₃⋅H₂O (cyclen = 1,4,7,10-tetraazacyclododecane) (2), [Co(NH₃)₅(OH₂)](CF₃SO₃)₃ (3) [Ni(NH₃)₆]Br₂ (4) and [Ru(NH₃)₆]Cl₃ (5) were tested against Sindbis infected baby hamster kidney (BHK) cells and show differential effects from the previously reported anti-viral complex [Co(NH₃)₆]Cl₃ (1). The macrocyclic complex 2 and labile aqua complex 3 show either no or little effect on the survival on Sindbis virus-infected cells as compared to that for 1, which show a monotonic increase in % BHK cell survival. Nickel and ruthenium ammine complexes 4 and 5 had a moderate influence of cell survival. While the results showed some anti-viral activity for some of the structural variations, it appears that 1, with its potential to be a broad-spectrum anti-viral compound, occupies a unique position in its ability to both significantly enhance cell survival and to decrease viral expression of infected cells. We also show that 1 also shows anti-viral activity against Adenovirus lending support to the broad-spectrum potential of this complex.     

NMR studies on the surface accessibility of the archaeal protein Sso7d by using TEMPOL and Gd(III)(DTPA-BMA) as paramagnetic probes

Understanding how proteins are approached by surrounding molecules is fundamental to increase our knowledge of life at atomic resolution. Here, the surface accessibility of a multifunctional small protein, the archaeal protein Sso7d from Sulfolobus solfataricus, has been investigated by using TEMPOL and Gd(III)(DTPA-BMA) as paramagnetic probes. The DNA binding domain of Sso7d appears very accessible both to TEMPOL and Gd(III)(DTPA-BMA). Differences in paramagnetic attenuation profiles of (1)H-(15)N HSQC protein backbone amide correlations, observed in the presence of the latter paramagnetic probes, are consistent with the hydrogen bond acceptor capability of the N-oxyl moiety of TEMPOL to surface exposed Sso7d amide groups. By using the gadolinium complex as a paramagnetic probe a better agreement between Sso7d structural features and attenuation profile is achieved. It is interesting to note that the protein P-loop region, in spite of the high surface exposure predicted by the available protein structures, is not approached by TEMPOL and only partially by Gd(III)(DTPA-BMA).     

In vitro studies on the DNA impairments induced by Cr(III) complexes with cellular reductants

The influence of Cr(III) complexes with ascorbic acid, cysteine and glutathione on DNA has been studied spectrophotometrically and chromatographically. The toxic and genotoxic activities of these complexes were also investigated. It was found that these complexes bind to DNA weaker than hexaaqua Cr(III) complexes. It could be explained through the greater strength of the bi- and tridentate ligands coordinated to chromium in comparison to water molecules. The formation of DNA-DNA intermolecular bonds and DNA interstrand cross-linking has been also observed. These complexes were found to be non-toxic and non-genotoxic in the bacterial test.     

Proteomic changes and molecular effects associated with Cr(III) and Cr(VI) treatments on germinating kiwifruit pollen

The present study is aimed at identifying molecular changes elicited by Cr(III) and Cr(VI) on germinating kiwifruit pollen. To address this question, comparative proteomic and DNA laddering analyses were performed. While no genotoxic effect was detected, a number of proteins whose accumulation levels were altered by treatments were identified. In particular, the upregulation of some proteins involved in the scavenging response, cell redox homeostasis and lipid synthesis could be interpreted as an oxidative stress response induced by Cr treatment. The strong reduction of two proteins involved in mitochondrial oxidative phosphorylation and a decline in ATP levels were also observed. The decrease of pollen energy availability could be one of the causes of the severe inhibition of the pollen germination observed upon exposure to both Cr(III) and Cr(VI). Finally, proteomic and biochemical data indicate proteasome impairment: the consequential accumulation of misfolded/damaged proteins could be an important molecular mechanism of Cr(III) toxicity in pollen.     

NMR studies on binary and ternary Pd(II) complexes formed by the growth-modulating tripeptide glycylhistidyllysine and nucleotides

The mechanism of transport of Pt(II) and Pd(II) into tissues through blood and that of their elimination in kidney is incompletely known so far. In this respect, the binding of palladium by the tripeptide glycyl-L-histidyl-L-lysine (GHL), a constituent of the human plasma, as a binary complex, and by the nucleotides 5'-IMP and 5'-GMP, as ternary complexes, has been studied by 1H and 13C NMR spectroscopy. These studies have been conducted in aqueous media and at different ligand/metal ratios. At acidic pH, resonances were observed for binary and ternary kinetically stable complexes, and binding sites in these complexes were identified by the effect of binding on chemical shifts of protons and carbon resonances. From these data, stoichiometries and structures of these complexes were proposed.     

Specific cleavage of DNA at CG sites by Co(III) and Ni(II) desferal complexes.

Hydrolysis of endothelin 1 by rat kidney membranes was investigated using a reverse-phase HPLC and an automated gas-phase protein sequencer. Endothelin 1 was hydrolyzed into four major fragments which were detected by HPLC. Phosphoramidon, an inhibitor of neutral endopeptidase 24,11, almost completely suppressed the production of three fragments, but one fragment was not affected by the inhibitor. Analysis of N-terminal sequences of the degradation products revealed that the phosphoramidon-sensitive fragments were generated by cleavage at the Ser5-Leu6 bond of endothelin 1 that was identical with its cleavage site by purified rat endopeptidase 24,11, reported previously. The phosphoramidon-insensitive fragment was produced by cleavage at Leu17-Asp18, which was distinct from the sites by endopeptidase 24,11, but corresponded to that by a phosphoramidon-insensitive metallo-endopeptidase recently isolated from rat kidney membranes by us [(1992) Eur. J. Biochem. 204, 547-552]. Kinetic determination of endothelin 1 hydrolysis by the isolated enzyme yielded values of Km = 71.5 microM and kcat = 1.49 s-1, giving a ratio of kcat/Km = 2.08 x 10(4) s-1.M-1. The Km value was much higher and the kcat/Km value was much lower than those for rat endopeptidase 24,11 reported previously. Thus, endopeptidase 24,11 appears to hydrolyze endothelin 1 more efficiently than the isolated enzyme does. Both enzymes may play physiological roles in the metabolism of endothelin 1 by rat kidney membranes in vivo.     

Investigations into the interaction between tumor-inhibiting ruthenium(III) complexes and nucleotides by capillary electrophoresis

Ruthenium(III) complexes of the general formula HL[RuCl₄L₂], with two trans-standing heterocyclic ligands L bound to ruthenium via nitrogen, show remarkable activity in different tumor models. To obtain a deeper insight into the mode of action of this class of anticancer compounds, we investigated the interaction of HIm trans-[RuCl₄(im)₂] (im, imidazole) and HInd trans-[RuCl₄(ind)₂] (ind, indazole) with all four nucleoside monophosphates in buffered solution by means of capillary electrophoresis. A preference for GMP- and AMP-coordination was found. A decrease of the pH resulted in a significantly increased amount of bound nucleotide. This feature seems to be interesting with regard to the lower pH values in solid tumors.     

NMR coalescence effects resulting from stereochemical non-rigidity and halide exchange in octahedral rhodium(III) and iridium(III) tertiary phosphine complexes

The PMe₂Ph ligands in the aquo-cations mer- [MCl₂(H₂O)(PMe₂Ph)₃][ClO₄] rapidly exchange on the NMR time-scale giving coalescence in the ¹H and ³¹P NMR spectra. Dissociation of the H₂O ligand which is trans to PMe₂Ph leads to a five- coordinate intermediate. This intermediate (M = Rh) is believed to be involved in the rapid reaction of [RhCl₂(H₂O)(PMe₂Ph)₃] [ClO₄] with mer- [RhCl₃(PMe₂Ph)₃] by a chloride transfer mechanism leading to total exchange of the PMe₂Ph ligands.     

Chemical cleavage of plasmid DNA by Cu(II), Ni(II) and Co(III) desferal complexes.

It is demonstrated that the Cu(II), Co(III) and Ni(II) complexes of a siderophore chelating drug desferal cleave DNA, in contrast to the corresponding Fe(II) complex which does not bring about DNA scission. Hydroxy radical scavengers inhibit the cleavage reaction.     

Prompt hydrolysis of adenosine 3', 5'-cyclic monophosphate by Co(III) complexes.

Adenosine 3',5'-cyclic phosphate (cAMP) is efficiently hydrolyzed at pH 7, 50 degrees C by use of [Co-(trien) (H2O)2]3+ and [Co(tme)2-(H2O)2]3+ complexes as catalysts: trien (diethylenetriamine) and tme (1,1,2,2-tetramethylethylenediamine). The acceleration is remarkable (10(8) to 10(9) fold), decreasing half-life of cAMP from 660,000 years to 7-15 hours.     

A Study of the Interaction of Cr(III) Complexes and Their Selective Binding with B-DNA: A Molecular Modeling Approach

Abstract

Molecular modeling and energy minimisation calculations have been used to investigate the interaction of chromium(III) complexes in different ligand environments with various sequences of B-DNA. The complexes are [Cr(salen)(H₂O)₂]⁺; salen denotes 1, 2 bis-salicylideneaminoethane, [Cr(salprn)(H₂O)₂]⁺; salprn denotes 1, 3 bis- salicylideneamino-propane, [Cr(phen)₃]³⁺; phen denotes 1, 10 phenanthroline and [Cr(en)₃]³⁺; en denotes eth- ylenediamine. All the chromium(III) complexes are interacted with the minor groove and major groove of d(AT)₁₂, d(CGCGAATTCGCG)₂ and d(GC)₁₂ sequences of DNA. The binding energy and hydrogen bond parameters of DNA-Cr complex adduct in both the groove have been determined using molecular mechanics approach. The binding energy and formation of hydrogen bonds between chromium(III) complex and DNA has shown that all complexes of chromium(III) prefer minor groove interaction as the favourable binding mode.     

Mononuclear and binuclear Co(III)-dioxygen adducts of bleomycin. Circular dichroism and electron paramagnetic resonance studies

Co(II) interacts with bleomycin in aqueous solution, in the presence of air, to give a short lived mononuclear superoxo-Co(III) complex (I) identified previously, by Sugiura, by electron paramagnetic resonance measurements. This complex rapidly releases O₂ to yield the dinuclear μ-peroxo-Co(III) complex (II), but is stabilized by the presence of DNA yielding a new superoxo long lived species (I′). The absorption and circular dichroism spectra of the three species (I,I′,II) have been characterized.