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Shao Y  Sheng X  Li Y  Jia ZL  Zhang JJ  Liu F  Lu GY 《Bioconjugate chemistry》2008,19(9):1840-1848
Novel cleft molecule pyridine-2,6-dicarboxamide appending two guanidinoethyl group side arms (Gua) was synthesized. The interactions of the cleft molecule in the absence of copper(II) (Gua) or in the presence of copper(II) (Cu2+-Gua) with calf thymus DNA were studied by fluorescence and CD spectroscopy. The results indicate that the DNA binding affinity of Cu2+-Gua is stronger than that of Gua, and the binding constants of Cu2+-Gua and Gua are 1.61 x 10(6) M(-1) and 2.86 x 10(5) M(-1), respectively. Agarose gel electrophoresis was used to assess the plasmid pUC 19 DNA cleavage activities in the presence of Gua and Cu2+-Gua. Kinetic data of DNA cleavage promoted by Cu2+-Gua under physiological conditions fit a saturation kinetic profile with k(max) of 0.0173 +/- 0.0011 h(-1), which gave a aproximately 10(6)-fold rate acceleration over uncatalyzed supercoiled DNA, while the catalyst concentration is lower than 0.0625 mM. The hydrolysis pathway was proposed as the possible mechanism for DNA cleavage promoted by Cu2+-Gua. The acceleration is due to efficient cooperative catalysis of the copper cation center and the functional groups (bis(guanidinium) groups).  相似文献   

3.
The mixed-ligand complexes [Cu(II)(HisLeu)(phen)](+) (1) and [Cu(II)(HisSer)(phen)](+) (2; phen=1,10-phenanthroline) were synthesized and characterized. The intercalative interaction of the Cu(II) complexes with calf-thymus DNA (CT-DNA) was probed by UV/VIS and fluorescence titration, as well as by thermal-denaturation experiments, and the intrinsic binding constants (K(b)) for the complexes with 1 and 2 were 4.2x10(3) and 4.9x10(3) M(-1), resp. Both complexes were found to be efficient catalysts for the hydrolytic cleavage of plasmid pUC19 DNA, as tested by gel electrophoresis, converting the DNA from the supercoiled to the nicked-circular form at rate constants of 1.32 and 1.40 h(-1) for 1 and 2, resp.  相似文献   

4.
The interactions of calf thymus DNA with tetracycline (TC), 7-chlorotetracycline (CTC) and 6-dimethyl-7-chlorotetracycline (DMTC) were assessed employing spectrofluorometric and circular dichroism (CD) techniques. The Scatchard analysis revealed relatively lesser binding affinity of TC (Ka= 1.2 x 10(7) lmol(-1)) vis-a-vis CTC (Ka= 3.4 x 10(7) lmol(-1)) and DMTC (Ka= 3.0 x 10(7) lmol(-1)) with DNA. The data suggested both the intercalative and electrostatic nature of binding between the tetracyclines and DNA. The presence of Cu(II) augmented the interaction of tetracyclines with DNA, and resulted in red shift by 12 nm in CD spectra of tetracycline. The molar ellipticity (theta) also changed significantly for CTC and DMTC. The data unequivocally demonstrated the DNA binding potential of tetracyclines both in the presence and absence of Cu(II) ions in dark. The enhanced binding of tetracyclines in presence of Cu(II), ensuing conformational changes in DNA secondary structure to a varying extent, reflects differential reactivity of ligand chromophores.  相似文献   

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6.
Electronic absorption spectra of flavomononucleotide (FMN) in poly(vinyl alcohol) films (PVA) were measured over the concentrations ranging from 6.9 x 10(-4) to 6.8 x 10(-1) M and temperatures from 263 to 338 K.The FMN absorption spectra measurements performed at room temperature have shown two ranges of different changes as a function of dye concentration. For concentrations c<10(-1) M (range I) the spectra exhibited regular changes showing an isosbestic point, which evidences the equilibrium between monomers and dimers. However, for range II (c>1.05 x 10(-1) M) the FMN absorption spectra occurred to be almost independent of concentration and they nearly overlapped with the dimer spectrum (within the error limit).Temperature measurements have shown that the FMN absorption spectra in PVA are stable over a wide temperature range.The mean distances between FMN molecules in PVA films are calculated. For maximal concentrations (from the range II), they are below 13.1 A, whereas the mean dimensions of FMN monomers and dimers are 15.8 and 21.1 A, respectively, which indicates that the orientation of dimers and monomers in the PVA film cannot be random at high concentrations. Molecules are partly ordered, adopting approximately parallel orientation, which is in agreement with the calculations of dimer structure by molecular modelling method (MMM).  相似文献   

7.
The copper (II) complex [Cu(Itpy)(2)](ClO(4))(2) (1), (Itpy=imidazole terpyridine) has been synthesized and structurally characterized. Crystal structure of the complex shows the complex to be a monomeric copper (II) species with two Itpy ligands coordinated to the metal ion to give a six coordinate complex. The complex has a distorted octahedral geometry with axial elongation. Variable temperature crystal structure data shows dynamic nature of the Jahn-Teller distortion. The complex is an avid DNA binder with a binding constant of 4.26+/-0.20x10(3)M(-1). Observed changes in the viscosity and circular dichroic spectrum of calf thymus DNA solution in the presence of complex 1 suggests intercalative binding of complex 1 to DNA. The complex cleaves supercoiled pBR322 DNA oxidatively in the presence of hydrogen peroxide.  相似文献   

8.
S(IV) (SO(2),HSO(3)(-)andSO(3)(2-)) autoxidation catalyzed by Cu(II)/tetraglycine complexes in the presence of DNA or 2'-deoxyguanosine (dGuo) resulted in DNA strand breaks and formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo), respectively. Ni(II), Co(II) or Mn(II) (1.0x10(-4)M) complexes had much smaller effects. Cu(II)/tetraglycine (1.0x10(-4)M) in the presence of Ni(II) or Mn(II) (10(-7)-10(-6)M) and S(IV) showed remarkable synergistic effect with these metal ions producing a higher yield of 8-oxodGuo. Oxidation of dGuo and DNA damage were attributed to oxysulfur radicals formed as intermediates in S(IV) autoxidation catalyzed by transition metal ions. SO*(3)(-) and HO* radicals were detected by EPR-spin trapping experiments with DMPO (5,5-dimethyl-1-pyrroline-N-oxide).  相似文献   

9.
The Sso10b (or Alba) family of proteins is a conserved group of archaeal and eukaryotic proteins which are thought to play a role in both chromatin organization and RNA metabolism. We describe here the solution structure and properties of Sso10b2 from Sulfolobus solfataricus. NMR data including residual dipolar couplings and (15)N relaxation data demonstrated that the protein adopts a beta(1)alpha(1)beta(2)alpha(2)beta(3)beta(4) topology with an IF-3-like fold. The protein dimerizes in solution at 30 degrees C via a hydrophobic surface defined by the C-terminal alpha(2)beta(3)beta(4) elements with a structure similar to one of the putative dimers indicated by previous crystal structures. DSC and circular dichroism data demonstrated an unusual two-state structural transition near the growth temperature which led to an increase in beta-sheet content without dissociation of the dimer. The cooperativity of the transition exceeded that of a dimer at pH 7, demonstrating the presence of higher order oligomers near the growth temperature at pH 7. Reverse titrations of Sso10b2 with nucleic acid showed that the protein binds single-stranded DNA (K(d) of 3 x 10(-)(7) M) with higher affinity than RNA (1.3 x 10(-)(6) M) or double-stranded DNA (1.5 x 10(-)(5) M) in 10 mM KH(2)PO(4) (pH 7.0, 20 degrees C). NMR chemical shift perturbation data indicated that single-stranded DNA and RNA binding occurred across the same dimer interface and encompassed a surface defined by the C-terminal ends of the beta(1), beta(2), and beta(3) strands of each monomer.  相似文献   

10.
1. The hemocyanin of the bivalve, Yoldia limatula (Say) was found by light-scattering to have a mol. wt of 8.0 +/- 0.6 x 10(6). Mass measurements by scanning transmission electron microscopy (STEM) gave a particle mass of 8.25 +/- 0.42 x 10(6) for the native particle and 4.09 +/- 0.20 x 10(6) for the half-molecule. 2. The hemocyanin subunits fully dissociated in 8.0 M urea and 6.0 M GdmCl at pH 8.0, and at pH 11.0, 0.01 M EDTA have mol. wts of 4.38 x 10(5), 4.22 x 10(5) and 4.71 x 10(5), close to one-twentieth of the parent molecular weight of Y. limatula hemocyanin and most gastropod hemocyanins. 3. Analyses of the urea dissociation transitions studied at pH 8.0, 1 x 10(-2) M Mg2+, 1 x 10(-2) M Ca2+ and pH 8.0, 3 x 10(-3) M Ca2+ suggest few hydrophobic amino acid groups, of the order of 10 to 15 at the contact areas of each half-molecule or decamer. 4. The further dissociation of the decamers to dimers and the dimers to monomers indicates the presence of a larger number of amino acid groups of ca 35-40/dimer and 100-120/monomer. 5. This suggests hydrophobic stabilization of the dimer to dimer and monomer to monomer contacts within the decamers, as observed with other molluscan hemocyanins.  相似文献   

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The coordination geometry around copper(II) in [Cu(imda)(phen)(H2O)] (1) (H2imda = iminodiacetic acid, phen = 1,10-phenanthroline) is described as distorted octahedral while those in [Cu(imda)(5,6-dmp)] (2) (5,6-dmp = 5,6-dimethyl-1,10-phenanthroline) and [Cu(imda)(dpq)] (3) (dpq = dipyrido-[3,2-d:2',3'-f]-quinoxaline) as trigonal bipyramidal distorted square-based pyramidal with the imda anion facially coordinated to copper(II). Absorption spectral (Kb: 1, 0.60+/-0.04x10(3); 2, 3.9+/-0.3x10(3); 3, 1.7+/-0.5x10(4) M(-1)) and thermal denaturation studies (deltaTm: 1, 5.70+/-0.05; 2, 5.5+/-10; 3, 10.6+/-10 degrees C) and viscosity measurements indicate that 3 interacts with calf thymus DNA more strongly than 1 and 2. The relative viscosities of DNA bound to 1 and 3 increase while that of DNA bound to 2 decreases indicating formation of kinks or bends and/or conversion of B to A conformation as revealed by the decrease in intensity of the helicity band in the circular dichroism spectrum of DNA. While 1 and 3 are bound to DNA through partial intercalation, respectively, of phen ring and the extended planar ring of dpq with DNA base stack, the complex 2 is involved in groove binding. All the complexes show cleavage of pBR322 supercoiled DNA in the presence of ascorbic acid with the cleavage efficiency varying in the order 3 > 1 > 2. The highest oxidative DNA cleavage of dpq complex is ascribed to its highest Cu(II)/Cu(I) redox potential. Oxidative cleavage studies using distamycin reveal minor groove binding for the dpq complex but a major groove binding for the phen and 5,6-dmp complexes. Also, all the complexes show hydrolytic DNA cleavage activity in the absence of light or a reducing agent with cleavage efficiency varying in the order 1 > 3 > 2.  相似文献   

13.
Staphylococcus aureus pI258 CadC is an extrachromosomally encoded metalloregulatory repressor protein from the ArsR superfamily which negatively regulates the expression of the cad operon in a metal-dependent fashion. The metalloregulatory hypothesis holds that direct binding of thiophilic divalent cations including Cd(II), Pb(II), and Zn(II) by CadC allosterically regulates the DNA binding activity of CadC to the cad operator/promoter (O/P). This report presents a detailed characterization of the metal binding and DNA binding properties of wild-type CadC. The results of analytical ultracentrifugation experiments suggest that both apo- and Cd(1)-CadC are stable or weakly dissociable homodimers characterized by a K(dimer) = 3.0 x 10(6) M(-1) (pH 7.0, 0.20 M NaCl, 25.0 degrees C) with little detectable effect of Cd(II) on the dimerization equilibrium. As determined by optical spectroscopy, the stoichiometry of Cd(II) and Pb(II) binding is approximately 0.7-0.8 mol/mol of wild-type CadC monomer. Chelator (EDTA) competition binding isotherms reveal that Cd(II) binds very tightly, with K(Cd) = 4.3 (+/-1.8) x 10(12) M(-1). The results of UV-Vis and X-ray absorption spectroscopy of the Cd(1) complex are consistent with a tetrathiolate (S(4)) complex formed by four cysteine ligands. The (113)Cd NMR spectrum reveals a single resonance of delta = 622 ppm, consistent with an S(3)(N,O) or unusual upfield-shifted S(4) complex. The Pb(II) complex reveals two prominent absorption bands at 350 nm (epsilon = 4000 M(-1) cm(-1)) and 250 nm (epsilon = 41 000 M(-1) cm(-1)), spectral properties consistent with three or four thiolate ligands to the Pb(II) ion. The change in the anisotropy of a fluorescein-labeled oligonucleotide containing the cad O/P upon binding CadC and analyzed using a dissociable CadC dimer binding model reveals that apo-CadC forms a high-affinity complex [K(a) = (1.1 +/- 0.3) x 10(9) M(-1); pH 7.0, 0.40 M NaCl, 25 degrees C], the affinity of which is reduced approximately 300-fold upon the binding of a single molar equivalent of Cd(II) or Pb(II). The implications of these findings on the mechanism of metalloregulation are discussed.  相似文献   

14.
Copper (II) complex 1 having planar tridentate ligand, bzimpy, where bzimpy is 2,6-bis(benzimidazo-2-yl) pyridine was synthesized and characterized by UV-visible, FAB (fast atom bombardment) mass and infrared spectroscopy. From absorption titration data, the binding constant of Cu(II) with DNA was calculated to be (1.8+/-0.02)x10(4) M(-1). Thermal denaturation study of DNA with 1 revealed deltaT(m) of 5+/-0.5 degrees C. Viscosity measurement showed that complex binds with DNA through intercalative mode. Copper (II) complex induces cleavage in plasmid DNA in the presence of coreductants such as ascorbic acid or glutathione.  相似文献   

15.
Arc repressor is tetrameric when bound to operator DNA   总被引:10,自引:0,他引:10  
B M Brown  J U Bowie  R T Sauer 《Biochemistry》1990,29(51):11189-11195
The Arc repressor of bacteriophage P22 is a member of a family of DNA-binding proteins that use N-terminal residues in a beta-sheet conformation for operator recognition. Here, Arc is shown to bind to its operator site as a tetramer. When mixtures of Arc (53 residues) and an active variant of Arc (78 residues) are used in gel retardation experiments, five discrete protein-DNA complexes are observed. This result is as expected for operators bearing heterotetramers containing 4:0, 3:1, 2:2, 1:3, and 0:4 ratios of the two proteins. Direct measurements of binding stoichiometry support the conclusion that Arc binds to a single 21-base-pair operator site as a tetramer. The Arc-operator binding reaction is highly cooperative (Hill constant = 3.5) and involves at least two coupled equilibria. In the first reaction, two unfolded monomers interact to form a folded dimer (Bowie & Sauer, 1989a). Rapid dilution experiments indicate that the Arc dimer is the kinetically significant DNA-binding species and allow an estimate of the equilibrium dissociation constant for dimerization [K1 = 5 (+/- 3) x 10(-9) M]. The rate of association of Arc-operator complexes shows the expected second-order dependence on the concentration of free Arc dimers, with k2 = 2.8 (+/- 0.7) x 10(18) M-2 s-1. The dissociation of Arc-operator complexes is a first-order process with k-2 = 1.6 (+/- 0.6) x 10(-4) s-1. The ratio of these kinetic constants [K2 = 5.7 (+/- 2.3) x 10(-23) M2] provides an estimate for the equilibrium constant for dissociation of the DNA-bound tetramer to two free Arc dimers and the operator. An independent determination of this complex equilibrium constant [K2 = 7.8 (+/- 4.8) x 10(-23) M2] was obtained from equilibrium binding experiments.  相似文献   

16.
Liu Y  Liu Y  Yang Z  Utzat C  Wang G  Basu AK  Zou Y 《Biochemistry》2005,44(19):7361-7368
Human xeroderma pigmentosum group A (XPA) is an essential protein for nucleotide excision repair (NER). We have previously reported that XPA forms a homodimer in the absence of DNA. However, what oligomeric forms of XPA are involved in DNA damage recognition and how the interaction occurs in terms of biochemical understanding remain unclear. Using the homogeneous XPA protein purified from baculovirus-infected Sf21 insect cells and the methods of gel mobility shift assays, gel filtration chromatography, and UV-cross-linking, we demonstrated that both monomeric and dimeric XPA bound to the DNA adduct of N-acetyl-2-aminofluorene (AAF), while showing little affinity for nondamaged DNA. The binding occurred in a sequential and protein concentration-dependent manner. At relatively low-protein concentrations, XPA formed a complex with DNA adduct as a monomer, while at the higher concentrations, an XPA dimer was involved in the specific binding. Results from fluorescence spectroscopic and competitive binding analyses indicated that the specific binding of XPA to the adduct was significantly facilitated and stabilized by the presence of the second XPA in a positive cooperative manner. This cooperative binding exhibited a Hill coefficient of 1.9 and the step binding constants of K(1) = 1.4 x 10(6) M(-)(1) and K(2) = 1.8 x 10(7) M(-)(1). When interaction of XPA and RPA with DNA was studied, even though binding of RPA-XPA complex to adducted DNA was observed, the presence of RPA had little effect on the overall binding efficiency. Our results suggest that the dominant form for XPA to efficiently bind to DNA damage is the XPA dimer. We hypothesized that the concentration-dependent formation of different types of XPA-damaged DNA complex may play a role in cellular regulation of XPA activity.  相似文献   

17.
The Escherichia coli Rep helicase is a stable monomer (Mr = 72,802) in the absence of DNA; however, binding of single-stranded (ss) or duplex (ds) DNA induces Rep monomers to dimerize. Furthermore, a chemically cross-linked Rep dimer retains both its DNA-dependent ATPase and helicase activities, suggesting that the functionally active Rep helicase is a dimer (Chao, K., and Lohman, T. M. (1991) J. Mol. Biol. 221, 1165-1181). Using a modified "double-filter" nitrocellulose filter binding assay, we have examined quantitatively the equilibrium binding of Rep to a series of ss-oligodeoxynucleotides, d(pN)n (8 less than or equal to n less than or equal to 20) and two 16-base pair duplex oligodeoxynucleotides, which are short enough so that only a single Rep monomer can bind to each oligonucleotide. This strategy has enabled us to examine the linkage between DNA binding and dimerization. We also present a statistical thermodynamic model to describe the DNA-induced Rep dimerization in the presence of ss- and/or ds-oligodeoxynucleotides. We observe quantitative agreement between this model and the experimental binding isotherms and have analyzed these isotherms to obtain the seven independent interaction constants that describe Rep-DNA binding and Rep dimerization. We find that Rep monomers (P) can bind either ss-DNA (S) or ds-DNA (D) to form PS or PD, respectively, which can then dimerize to form P2S or P2D. Furthermore, both protomers of the DNA-induced Rep dimer can bind DNA to form either P2S2, P2D2 or the mixed dimer species P2SD and ss- and ds-DNA compete for the same sites on the Rep protein. When bound to DNA, the Rep dimerization constants are approximately 1-2 x 10(8) M-1 (6 mM NaCl, pH 7.5, 4 degrees C), which are greater than the dimerization constant for free Rep monomers by at least 10(4)-fold. The Rep-ss-DNA interaction constants are independent of base composition and sequence, consistent with its role as a nonspecific DNA-binding protein. Allosteric effects are associated with ss- and ds-DNA binding to the half-saturated Rep dimers, i.e. the affinity of either ss- or ds-DNA to the free promoter of a half-saturated Rep dimer is clearly influenced by the conformation of DNA bound to the first protomer. These allosteric effects further support the proposal that the Rep dimer is functionally important and that the Rep-DNA species P2S2 and P2SD may serve as useful models for intermediates that occur during DNA unwinding.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

18.
Behavior of topotecan, DNA topoisomerase I inhibitor, was studied in aqueous solutions by optical methods. Topotecan absorption spectra were recorded in the pH range 0.5-11.5 and its pKa were determined. Quantum chemical calculations were made for all charge states of the topotecan molecule in lactone and carboxylate form. The calculated absorption maxima agree well with the experimental data. Protonation of the topotecan D ring (pKa = 3.6) was revealed. Comparison of experimental and calculated data showed topotecan structure with a proton at the oxygen atom at C16a rather than N4 to be the most preferable. Topotecan molecules were shown to form dimers at concentrations above 10(-5) M. Topotecan dimerization is accompanied by an increase in the pKa of hydroxy group of the A ring from 6.5 ([TPT] = 10(-6) M) to 7.1 ([TPT] = 10(-4) M), which indicates participation of this group in dimer stabilization, perhaps due to intermolecular hydrogen bonding with N1 of the B ring of a neighboring molecule. Probable dimer structures were proposed. The topotecan dimerization constant was determined, K = (4.0 +/- 0.7) x 10(3) M-1.  相似文献   

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20.
The effects of HU, the histone-like protein from Escherichia coli, on the equilibrium cyclization of duplex DNAs have been observed as a function of protein concentration and DNA sequence. The results indicate that the presence of HU significantly enhances the extent of cyclization and increases the melting temperature, T(m), of the cyclized form of the DNA by >10 K. The stabilization of equilibrium cyclization by HU binding is at least -1.2 kcal/mol. The results are consistent with two HU homotypic dimers binding to each of the three 29-mer duplexes studied. One of the 29-mer duplexes contains a central dA tract, one contains mismatched sites, and one a conventional sequence. Stepwise or microscopic association constants, determined from the fluorescence data, range from 1.5 to 0.6 micro M(-1). The binding affinity of the HU dimer is strongest for the mismatched duplex and lowest for the dA tract, consistent with HU dimers having a preference for flexible DNA substrates. These results demonstrate the utility of the equilibrium cyclization approach to monitor DNA-protein interactions. These results have been considered along with those previously obtained to refine a model for the interaction of HU with duplex DNA.  相似文献   

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