Phase partition studies of actinomycin-nucleotide complexes.

A method is reported for measuring the stoichiometry of complex formation between actinomycin and a series of deoxynucleotides. The amount of bound actinomycin is measured by distribution of the drug between two liquid phases, a buffer phase containing deoxynucleotide and an organic phase in which the nucleotide is insoluble. Using simple statistical mechanical analysis, the equilibrium equations for several models of actinomycin-deoxynucleotide complexes have been derived: actinomycin with one binding site, with two equivalent independent binding sites, and with two sites which must be occupied together. The binding of actinomycin C3 with dpG, dpApG, dpA, and dpGpC has been examined compared with these models. It is found that binding to dpG and dpApG involves two independent binding sites of nearly equal affinity for nucleotides, whereas binding of dpGpC to the two binding sites on actinomycin is a cooperative process. Binding of dpA tp actinomycin is partially cooperative and weaker than binding of dpG. The dimerization constant of actinomycin was also determined by the phase separation technique, and found in agreement with other values, including the results of kinetic measurements reported here.     

Actinomycin D complexes with oligonucleotides as models for the binding of the drug to DNA. Paramagnetic induced relaxation experiments on drug-nucleic acid complexes.

Mn(II) ions have been used as a paramagnetic probe to investigate the geometry of drug-oligonucleotide complexes. Nuclear magnetic resonance and electron spin resonance experiments show that Mn(II) ions bind approximately two orders of magnitude stronger to the 5'-terminal phosphate group than to the 3'-5' phosphodiester linkage of deoxydinucleotides. By using mixtures of nucleotides in which only one nucleotide contains a terminal phosphate group, the location of the Mn(II) ion in the drug-nucleotide-Mn(II) complexes may be preselected. The paramagnetic induced relaxation of the nuclear spin systems in these complexes has been used to investigate the geometry of these complexes. These data confirm that actinomycin D is able to recognize and preferentially bind guanine (as opposed to adenine) nucleotides in the quinoid portion of the phenoxazone ring, while both adenine and guanine will bind to the benzenoid portion of the phenoxazone ring. These results suggest that stacking forces are primarily responsible for the general requirement of a guanine base when actinomycin D binds to DNA.     

Two-dimensional NMR spectroscopy of siomycin A. Proton--carbon-13 chemical shift correlation

A trial application of a recent two-dimensional nuclear magnetic resonance experiment to the polypeptide antibiotic siomycin A is described. Proton--carbon-13 chemical shift correlation measures the proton and carbon-13 chemical shift for each directly bonded CH group in a molecule, in a single experiment. The resultant map of correlated chemical shifts enables the carbon-13 spectrum to be assigned directly from the known proton shifts, and allows individual proton signals to be identified without problems of overlap. The signal-to-noise ratio available from such techniques should enable their application to aqueous protein solutions using currently available high-field spectrometers.     

Effects of pD on ring-stacking interactions between dinucleoside phosphates and tryptamine

Complex formation between tryptamine and mononucleotides and dinucleoside phosphates containing adenine and/or cytosine has been studied at five pD's ranging from 1.1 to 7.4 by proton magnetic resonance spectroscopy. Chemical shifts of base ring protons and the ribose anomeric proton in the nucleotides and indole ring protons in tryptamine were monitored and their changes with pD and intermolecular interactions interpreted qualitatively. Stacked complexes were found to exist at all pD's in the range studied. Complex geometries differ depending on pD. An electrostatic interaction between the tryptamine amino group and the nucleotide phosphate group contributes to complex formation above pD 4 but is not strong enough to shift the dinucleoside phosphate equilibrium towards the unstacked conformer.     

Assignment of the 13C nuclear magnetic resonance spectrum of a short DNA-duplex with 1H-detected two-dimensional heteronuclear correlation spectroscopy.

Proton-detected 1H-13C heteronuclear correlated spectroscopy [( 1H,13C]-COSY) was used to establish relations between the carbon-13 and proton nuclear magnetic resonance chemical shifts in the hexadeoxynucleoside pentaphosphate d-(GCATGC)2. Using the previously established sequence-specific proton NMR assignments, sequence-specific assignments were thus obtained for nearly all proton-bearing carbons. This approach offers a new criterion for distinguishing between the proton NMR lines of purines and pyrimidines, based on the different proton-carbon-13 coupling constants. Furthermore, the adenine ring carbon 2 has a unique carbon-13 chemical shift, which enables a straightforward identification of the adenine C2H resonances by [1H,13C]-COSY.     

1H-NMR analysis of heteroassociation of caffeine with antibiotic actinomycin D in the aqueous solution

The molecular basis of the action of caffeine as a complex forming agent, an interceptor of aromatic drugs intercalating into DNA was studied by the example of the an anticancer antibiotic actinomycin D examined. The hetero-association of caffeine and actionomycin D was studied by one- and two-dimensional 1H-NMR spectroscopy (500 MHz). Concentration and temperature dependences of the proton chemical shifts of molecules in aqueous solution were measured. The equilibrium reaction constant of hetero-association of caffeine with actinomycin D (K = 246 +/- 48 M-1), the limiting chemical shifts of caffeine protons in complexes were determined. The most favourable structure of the 1:1 caffeine-actinomycin D hetero-complex in aqueous solution was constructed using the calculated values of the induced proton chemical shifts of molecules and the quantum-mechanical iso-shielding curves for caffeine and actinomycin D. The thermo-dynamical parameters of the hetero-complex formation between caffeine and actinomycin D were also determined. The structural and thermo-dynamical analysis showed that dispersive forces and hydrophobic interactions play the major role in hetero-association of caffeine and actinomycin D in aqueous-salt solution. The relative content of different complexes in mixed solutions containing caffeine and actinomycin D was calculated and distinctive features of the dynamic equilibrium of caffeine-actinomycin D hetero-associates were revealed as a function of concentration and temperature. It is concluded that hetero-association of caffeine and actinomycin D molecules a lowers the effective concentration of the drug in solution and hence the pharmacological activity of actinomycin D.     

Stereochemistry of actinomycin binding to DNA : II. Detailed molecular model of actinomycin-DNA complex and its implications

The three-dimensional structure of a crystalline complex containing actinomycin D and deoxyguanosine (described in the previous paper) has shed light on the stereochemistry of actinomycin binding to DNA. The phenoxazone ring system on actinomycin intercalates between the base-paired dinucleotide sequence, GpC, while the peptide subunits lie in the narrow groove of the DNA helix and interact with deoxyguanosine residues on opposite chains through specific hydrogen bonds. The binding of actinomycin to DNA demonstrates a general principle which several classes of proteins may utilize in recognizing symmetrically arranged nucleotide sequences on the DNA helix.     

Coordination mode and oxidation susceptibility of nickel(II) complexes with 2'-deoxyguanosine 5'-monophosphate and l-histidine

The formation of binary and ternary complexes of Ni(II) with two biologically relevant molecules, 2'-deoxyguanosine 5'-monophosphate (dGMP) and l-histidine (histidine or His) was characterized by potentiometry and UV-visible spectroscopy. For dGMP, the mononuclear complexes with stoichiometries NiH(2)L(+), NiHL and NiL(-) were found. In the mixed system the ternary complexes NiH(2)LA, NiHLA(-) and NiLA(2-) were detected. In binary systems, the Ni(II) ion coordinates to dGMP through the N-7 atom of its purine ring and indirectly through a water molecule bonded to the phosphate group, while in ternary complexes Ni(II) is bonded to all three histidine donors and directly to the phosphate group of dGMP. Both binary and ternary complexes are susceptible to oxidation by H(2)O(2), with the increased formation of 8-oxo-dGMP in the ternary system. The toxicological relevance of these findings stems from possible disturbance by the major biological Ni(II)-His complex of the nucleotide pools homeostasis through the formation of ternary species and oxidation promotion, as well as from 8-oxo-dGMP capacity to inhibit enzymatic elimination of promutagenic oxidized nucleotides from such pools.     

Carbon-13 magnetic resonance spectra of nucleosides and their Pd(II) complexes.

Chemical shifts occurring in carbon-13 magnetic resonance spectroscopy are utilized to assess the site of complexation of nucleosides to enPdC12 in neutral aqueous solutions. Binding occurs at N3 in cytidine, thymidine, and uridien, at N7 in 1-methylguanosine, and at N1 in guanosine. For most carbon atoms adjacent to N3 in the pyrimidine nucleosides the complexation shifts of the basic ligand are about 30% of the corresponding upfield protonation shifts. All complexes are of the form enPdL2 indicating that the ligands are unidentate and that the tendency to chelation is weak. Carbon-13 magnetic resonance spectroscopy appears to be the best method for delineating these complexes in solution. Due to the high avidity of chloride ion for Pt(II), cis dichloro Pd(II) complexes may be better models for intracellular action of the corresponding Pt(II) complexes than the Pt(II) complexes themselves.     

Resolution of electronic absorption bands and nucleotide binding processes in actinomycin D

Complexes of actinomycin D with model dexoxynucleoides have been studied by means of absorption spectroscopy and CD spectroscopy and CD spectroscopy. The CD spectra of the complexes of actinomycin D with 5′-dGMP, pdG-dT, pdT-dG, pdG-dA, and pdA-dG, respectively, all resemble one another. It is presumed that in solution the interactions between the guanine residues and actinomycin D in these complexes are the same as found in the crystalling 1:2 actinomycin D:dG complex [Jain, S. C. & Sobell, H. M. (1972) J. Mol. Biol. 68 , 1–20]. The CD spectrum of the Complexes with pdG-dC differs from of the complexes just mentioned, and resembles those of the complexes formed by actinomycin D with calf-thymus DNA and with poly(dG-dC)-poly(dG-dC). These resulls are consisent with, the nontion that pdG-dC froms a double-staranded intercalated complex with actinomycin D, and that the dG-dC sequence is an important binding site for actinomycin D in polydeoxynucleotides. Titrations of actinomycin D with monodeoxynucleotides were monitored at 380, 425, 440,465, and 480, nm in both absorption and CD modes. Comparisons fo saturation profiles at these wavelengths reveal that the curves obtained at various wavelenths do not superimpose with each other, so that they must reflect different titation processes. In complex formation wiht any given nucleotide, the apparent binding affinity monitored at these wavelengths decreases in the order given above. Based on these resulted and on features noted in the CD spectra of certain complexes, it is concluded that a minimum of theree electronic transitions underlie the visible absorption band of actinomycin D, extending earlier findigs. Comparing the titration proffiles obtained with dAMP and dIMP with the result for these systems in mmr titratins [Krugh, T. R. & Chen, Y. C. (1975) Biochemistry 14 , 4912–4922], it is concluded that one transition, centered at 370 nm, monitors preponderantly effects occurring at the 6 (benzenoid) nucleotide binding site and a second transition, located at 490 nm, is sensitive preferentially to processes occurring at the 4 ( quinoid) binding site. The latter is probably closely asscoiated with 2-amino and /or 3-carbonyl substituents. The third transition, identified with the absorption maxium at 420–440 nm, appears to reflect contributions arising in the entire phenoxazone chromophore. Using these band assignments, it is concluded that the benzenoid site binds nucleotides 1.5–3 times more avidly than does the quinoid site. CD titrations resolve these processes more effectively than do abscrption titrations. Aspects of the structures of these complexes formed in solution are discussed.     

NMR investigation of the complexation of daunomycin with deoxytetranucleotides of different base sequence in aqueous solution

500 MHz NMR spectroscopy has been used to investigate the complexation of the anthracycline antibiotic daunomycin (DAU) with self-complementary deoxytetranucleotides, 5'-d(CGCG), 5'-d(GCGC), 5'-d(TGCA), 5'-d(ACGT) and 5'-d(AGCT), of different base sequence in aqueous salt solution. 2D homonuclear 1H NMR spectroscopy (TOCSY and NOESY) and heteronuclear 1H - 31P NMR spectroscopy (HMBC) have been used for complete assignment of the non-exchangeable protons and the phosphorus resonance signals, respectively, and for a qualitative determination of the preferred binding sites of the drug. Analysis shows that DAU intercalates preferentially into the terminal sites of each of the tetranucleotides and that the aminosugar of the antibiotic is situated in the minor groove of the tetramer duplex, partly eclipsing the third base pair. A quantitative determination of the complexation of DAU with the deoxytetranucleotides has been made using the experimental concentration and temperature dependences of the drug proton chemical shifts; these have been analysed in terms of the equilibrium reaction constants, limiting proton chemical shifts and thermodynamical parameters (enthalpies deltaH, entropies deltaS) of different drug-DNA complexes (1:1, 1:2, 2:1, 2:2) in aqueous solution. It is found that DAU interacts with sites containing three adjacent base pairs but does not show any significant sequence specificity of binding with either single or double-stranded tetranucleotides, in contrast with other intercalating drugs such as proflavine, ethidium bromide and actinomycin D. The most favourable structures of the 1:2 complexes have been derived from the induced limiting proton chemical shifts of the drug in the intercalated complexes with the tetranucleotide duplex, in conjunction with 2D NOE data. It has been found that the conformational parameters of the double helix and the orientation of the DAU chromophore in the intercalated complexes depend on base sequence at the binding site of the tetramer duplexes in aqueous solution.     

1H and 31P NMR investigations of actinomycin D binding selectivity with oligodeoxyribonucleotides containing multiple adjacent d(GC) sites

Imino proton and 31P NMR studies were conducted on the binding of actinomycin D (ActD) to self-complementary oligodeoxyribonucleotides with adjacent 5'-GC-3' sites. ActD showed very high specificity for binding to GC sites regardless of oligomer length and surrounding sequence. For a first class of duplexes with a central GCGC sequence, a mixture of 1:1 complexes was observed due to the two different orientations of the ActD phenoxazone ring system. Analysis of 1H chemical shifts suggested that the favored 1:1 complex had the benzenoid side of the phenoxazone ring over the G base in the central base pair of the GCGC sequence. This is the first case in which an unsymmetrical intercalator has been shown to bind to DNA in both possible orientations. A unique 2:1 complex, with significantly different 1H and 31P chemical shifts relative to those of the 1:1 complexes, was formed with these same oligomers, again with the benzenoid side of the ActD molecule over the G base of the central GC base pair. There is considerable anticooperativity to binding of the second ActD in a GCGC sequence. In titrations of oligomers with the GCGC sequence, only the two 1:1 complexes are found up to ratios of one ActD per oligomer. Increasing the ActD concentration, however, resulted in stoichiometric formation of the unique 2:1 adduct. Spectrophotometric binding studies indicated that the apparent binding equilibrium constant for a GC site adjacent to a bound site is reduced by approximately a factor of 20 relative to the ActD binding constant to an isolated GC site.     

Hydrogen-bonded complexes of the ribodinucleoside monophosphates in aqueous solution. Proton magnetic resonance studies.

A proton magnetic resonance study of the chemical shifts of a series of ribodinucleoside monophosphates in neutral H2O solution has been recorded in the 1-100 mM concentration range. The self-complementary dinucleoside monophosphates CpG and GpC and the complementary mixture GpU + ApC form intermolecular hydrogen-bonded complexes at low temperatures. The amino proton chemical shifts in the CpG and GpC spectra are consistent with the formation of a miniature double helical dimer in neutral aqueous solution at low temperatures (approximately 2 degrees C). The complementary mixture of dinucleosides GpU + ApC formed much less stable complexes than either GpC or CpG, while UpA did not show any indication of the formation of intermolecular hydrogen-bonded complexes. This result is consistent with the well-known observation that the stability of a double helix is proportional to the percent of G-C base pairs present.     

Platinum complexes of nucleotides.

The reaction of [Pt(dien)Cl1Cl (dien = NH2CH2CH2NHCH2CH2NH2) with nucleotides has been studied by nuclear magnetic resonance. It has been found that the CMP (cytidine 5'-monophosp-ate) and GMP (guanosine 5'-monophosphate/coordinate to the platinum atom through N3 and N7, respectively. The reaction of the platinum salt with the nucleotide is complete when one to one ratio of platinum to nucleotide is used and no evidence of phosphate group binding to platinum has been found. No additional binding sites have been detected except the N7 site on the guanylic group of GMP even in the presence of a large excess of [Pt(dien) Cl1Cl. The AMP (adenosine 5'monophosphate] coordinates to the platinum at the N1 and/or N7 sites. The reaction of AMP and platinum is complete is complete at a ratio of four platinum to one AMP.     

Carbon-13 nuclear magnetic resonance of heme carbonyls. Cytochrome c and carboxymethyl derivatives of cytochrome c.

Carbonyl complexes of horse cytochrome c and various carboxymethylated derivatives have been examined using 13C NMR (carbon-13 nuclear magnetic resonance) spectroscopy. The multiplicity and chemical shift of the 13CO resonance were found to be functions of pH and the extent of alkylation. Correlations have been made among prominent features of the chemical shift titration curves and changes in the environment of the heme. A simple model compatible with the bulk of previous observations has been suggested to account for the several carbonyl resonance peaks and the complex behavior of the chemical shift with changes in pH.     

Studies on the conformation and interactions of elastin secondary structure of synthetic repeat hexapeptides.

Repeat hexapeptides of elastin have been synthesized and studied with nuclear magnetic resonance methods. The deuterium substituted hexapeptide HCO-Ala1-Pro2-(2H2) Gly3-Val4-Gly5-Val6-OMe allowed completion of the proton assignments and specifically the definitive assignments of the Gly3 and Gly5 resonances. Solvent titrations followed by carbon-13 magnetic resonances are reported which delineate the Ala1 C-O and Gly5 C-O as intramolecularly hydrogen bonded. This coupled with the proton magnetic resonance data which delineated the Gly3 NH and VAL4 NH as candidates for intramolecular hydrogen bonding lead to the proposal of two hydrogen bonds, one between the Ala1 C-O and the Val4NH and the second between the Gly5C-O and the Gly3NH. The probability, or mol fraction, of occurrence of these secondary structural features is demonstrated to be high.     

Studies on the conformation and interactions of elastin: nuclear magnetic resonance of the polyhexapeptide.

Synthesis, proton magnetic resonance and carbon-13 magnetic resonance characterizations, including complete assignments, are reported for the polyhexapeptide of elastin, HCO-Val(Ala1-Pro2-Gly3-Val4-Gly5-Val6)18-OMe. Temperature dependence of peptide NH chemical shifts and solvent dependence of peptide C-O chemical shifts have been determined in several solvents and have been interpreted in terms of four hydrogen bonded rings for each repeat of the polyhexapeptide. The more stable hydrogen bonded ring is a beta-turn involving Ala1C-O--HN-Val4. More dynamic hydrogen bonds are an 11-atom hydrogen bonded ring Gly3NH--O-C Gly5, a 7-atom hydrogen bonded ring (a gamma-turn) Gly3 C-O--NH-Gly5, and a 23-atom hydrogen bonded ring Val6inH--O-C Val6(i+1). This set of hydrogen bonds results in a right-handed beta-spiral structure with slightly more than two repeats (approximately 2.2) per turn of spiral. The beta-spiral structure is briefly discussed relative to data on the elastic fiber.     

Nucleotide and AP5A complexes of porcine adenylate kinase: A 1H and 19F NMR study

Proton and fluorine nuclear magnetic resonance spectroscopies (NMR) were used as methods to investigate binary complexes between porcine adenylate kinase (AK1) and its substrates. We also studied the interaction of fluorinated substrate analogues and the supposed bisubstrate analogue P1,P5-bis(5'-adenosyl) pentaphosphate (AP5A) with AK1 in the presence of Mg2+. The chemical shifts of the C8-H, C2-H, and ribose C1'-H resonances of both adenosine units in stoichiometric complexes of AK1 with AP5A in the presence of Mg2+ could be determined. The C2-H resonance of one of the adenine bases experiences a downfield shift of about 0.8 ppm on binding to the enzyme. The chemical shift of the His36 imidazole C2-H was changed in the downfield direction on ATP-Mg2+ and, to a lesser extent, AMP binding. 19F NMR chemical shifts of 9-(3-fluoro-3-deoxy-beta-D-xylofuranosyl)adenine triphosphate (3'-F-X-ATP)-Mg2+ and 9-(3-fluoro-3-deoxy-beta-D-xylofuranosyl)adenine monophosphate (3'-F-X-AMP) bound to porcine adenylate kinase could be determined. The different chemical shifts of the bound nucleotides suggest that their mode of binding is different. Free and bound 3'-F-X-AMP are in fast exchange with respect to their 19F chemical shifts, whereas free and bound 3'-F-X-ATP are in slow exchange on the NMR time scale in the absence as well as in the presence of Mg2+. This information could be used to determine the apparent dissociation constants of the nucleotides and the 3'-F-X analogues in the binary complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton magnetic resonance studies of double helical oligonucleotides. The effect of base sequence on the stability of deoxydinucleotide dimers.

The concentration dependence of the proton magnetic resonance chemical shifts of a series of deoxydinucleotides and deoxydinucleoside monophosphates in neutral H2O solution has been recorded in the 1-100 mM concentration range by the use of pulsed Fourier transform techniques. The self-complementary molecules pdG-dC, dG-dC, pdC-dG, and dC-dG and the complementary mixtures pdG-dG + pdC-dC as well as pdG-dT + pdA-dC interact at low temperatures by the formation of intermolecular hydrogen bonded dimers. Noncomplementary molecules such as pdG-dT, pdT-dG, pdG-dG, pdA-dc, and pdC-dC do not self-associate by the formation of intermolecular hydrogen bonds under the present experimental conditions. The chemical shifts of the amino protons and the base protons are consistent with the interaction of two complementary dinucleotides to form a miniature double helix. An analysis of the chemical shift of the guanine amino proton resonance as a function of dinucleotide concentration has provided approximate dimerization constants. These results show that the stability of the miniature double helices is in the order (pdG-dG)-(pdC-dC) greater than or approximately (pdG-dC)-(pdG-dC) greater than (pdC-dG)-(pdC-dG) greater than (pdG-dT)-(pdA-dC) which reflects the effect of nucleotide sequence (and composition) on helix stability.