Sodium-23 NMR spin-lattice relaxation rate studies of mono- and bis-intercalation in DNA

23Na spin-lattice relaxation rate (1/T1 = R1) measurements have been used to study the intercalation of a series of 9-aminoacridine derivatives in DNA. The 23Na relaxation rate is strongly dependent upon the amount of intercalator added to a sodium DNA solution. The results are analyzed by a combined use of the ion condensation theory and the quadrupolar relaxation theory of polyelectrolyte solutions. This interpretation shows that the major effect in lowering the relaxation rate by intercalation is not due to the release of sodium ions but is caused by a substantial decrease in the relaxation rate Rb for the remaining bound sodium ions. Likewise, titration of NaDNA solutions with MgCl2 shows that condensation of Mg2+ on the DNA double helix reduces Rb. A good agreement between experiment and theory is found if the average lengthening following intercalation of a 9-aminoacridine moiety is assumed to be approximately 2.7 A. The distinction between mono- and bis-intercalation is clearly indicated by the results. The two bis-intercalating drugs examined are found to bis-intercalate only up to r less than or equal to 0.02. For r greater than 0.02 the drugs apparently mono-intercalate.     

Binding of 9-aminoacridine to deoxydinucleoside phosphates of defined sequence: Preferences and stereochemistry

The effect of sequence on the binding of 9-aminoacridine to DNA has been investigated by studying its interaction with deoxydinucleoside phosphates of different sequences using proton nuclear magnetic resonance. Quantitative binding information can be obtained by comparison of the proton chemical shift behavior of 9-aminoacridine upon addition of dinucleoside phosphate to various models for the interaction using least-squares computer fitting procedures. The simplest model that fits the data includes (1) dimerization of 9-aminoacridine and (2) a mixture of 1:1 and 2:1 (dinucleoside phosphate/9-aminoacridine) complexes. The computed parameters allow comparison of binding constants and stereochemistry for different sequences. The 1:1 complexes seem to involve interaction of the ring nitrogen with the backbone phosphate and stacking of one or both chromophores on the acridine; preference in binding is observed for alternating (purine-pyrimidine or pyrimidine-purine) over non-alternating (purine-purine) dinucleoside phosphates. The 2:1 complexes involve intercalation of the acridine between two complementary dinucleoside phosphate strands with weak sequence preferences in binding. The stereochemistry of intercalation differs between non-alternating purine-purine sequences and the alternating pyrimidine-purine or purine-pyrimidine sequences in having the 9-aminoacridine stacked with the purines of one strand rather than straddling the purines on opposite strands. The difference in stereochemistry could possibly be a determining factor in frameshift sequence specificity.     

Nuclear magnetic relaxation studies of cytidine and its complexing dimethyl sulfoxide solution.

13C spin-lattice relazation times (T1 values) in cytidine were determined experimentally to investigate molecular motions of both metal-free and ion-complexed cytidines in dimethylsulfoxide solutions. It was found that the correlation times of the protonated carbons were equal within experimental error, and this equality of correlation times of different sites of the molecule suggests strongly isotropic random motion of the molecule. Correlation times for internal motion of the amino group obtained from the observed T1 of the amino protons are 4.6 -10(-11) S, 2.0 - 10(-9) S and 1.1 - 10(-10) S for the metal-free cytidine and the cytidine complexed with either CaCl2 or ZnCl2, respectively. An experimental value of T1 of the H6 proton of the cytidine base agrees very well with the value estimated from a conformation determined by the nuclear "Overhauser" effect. Spin-lattice relaxation time measurements of the 7Li nucleus in the LiCl/cytidine system strongly suggested that the 7Li cation is directly coordinated with cytidine.     

The interaction of an anti-tumour platinum complex with DNA.

The interaction of the anti-tumour active cis platinum (II) complexes with DNA has been investigated using dichloro(ethylenediamine)platinum(II) and E. coli DNA. Equilibrium dialysis studies indicate that Pt(en)Cl2 binds reversibly to DNA to a saturation value of 0.57 Pt: P, which is consistent with the platinum being bound both monofunctionally and bifunctionally. Pt(en)Cl2 inhibits the intercalation of 9-aminoacridine (9AA) by cross-linking the bases of the double helix, but at no stage does all the bound platinum cross-link. It is suggested that this inhibition of intercalation is due to intrastrand cross-linking.     

Impulse NMR study of the mobility of water adsorbed onto cellulose

An impulse method of nuclear magnetic resonance was used for measuring the times of spin-lattice relaxation in the rotating system of coordinates (RSC) for water molecules adsorbed on cottone cellulose. It has been shown that within the temperature region -10 divided by -40 degrees C the spin-lattice relaxation of water in RSC is conditioned by intermolecular interactions modulated with translation movement. The selfdiffusion coefficient of adsorbed water for the sample with 55% humidity at -10 degrees C is determined as 2.0.10(-9) cm2/s and decreases to 0.3.10(-9) cm2s at -40 degrees C, with activation energy of diffusion equalling 8.1 kcal/mol.     

Internal motions in B- and Z-form poly(dG-dC).poly(dG-dC): 1H NMR relaxation studies

Proton NMR relaxation measurements are used to compare the molecular dynamics of 60 base pair duplexes of B- and Z-form poly(dG-dC).poly(dG-dC). The relaxation rates of the exchangeable guanine imino protons (Gim) in H2O and in 90% D2O show that below 20 degrees C spin-lattice relaxation is exclusively from proton-proton magnetic dipolar interactions while proton-nitrogen interactions contribute about 30% to the spin-spin relaxation. The observation that the spin-lattice relaxation is nonexponential and that the initial spin-lattice relaxation rate of the Gim, G-H8 and C-H6 protons depends on the selectivity of the exciting pulse shows that spin-diffusion dominates the spin-lattice relaxation. The relaxation rates of the Gim, C-H5, and C-H6 in B- and Z-form poly(dG-dC).poly(dG-dC) cannot be explained by assuming the DNA behaves as a rigid rod. The data can be fit by assuming large-amplitude out of plane motions (+/- 30-40 degrees, tau = 1-100 ns) and fast, large-amplitude local torsional motions (+/- 25-90 degrees, tau = 0.1-1.5 ns) in addition to collective torsional motions. The results for the B and Z forms show that the rapid internal motions are similar and large in both conformations although backbone motions are slightly slower, or of lower amplitude, in Z DNA. At high temperatures (greater than 60 degrees C), imino proton exchange with solvent dominates the spin-lattice relaxation of B-form poly(dG-dC).poly(dG-dC), but in the Z form no exchange contribution (less than 2 s-1) is observed at temperatures as high as 85 degrees C. Conformational fluctuations that expose the imino protons to the solvent are strikingly different in the B and Z forms. The results obtained here are compared with those previously reported for poly(dA-dT).poly(dA-dT).     

A study of the quadrupolar NMR splittings of 7Li+, 23Na+, and 133Cs+ counterions in macroscopically oriented DNA fibers.

The hydration and temperature dependencies of the 23Na+, 133Cs+, and 7Li+ quadrupolar splitting have been determined in hydrated, macroscopically oriented DNA fibers. At low water contents the quadrupolar splitting is found to decrease as the water content increases, regardless of counterion, while at high water contents the hydration dependence is reversed. The 23Na+ and 133Cs+ quadrupolar splittings decrease as the temperature increases, while the 7Li+ splitting shows the opposite behavior. At high water contents the 23Na+ and 133Cs+ splittings decrease, and then, after passing zero splitting, increase as the temperature increases. The interpretation of the temperature dependence is discussed in terms of a two-site model (free and bound ions) and a three-site model (free ions and specifically or nonspecifically bound ions). It is suggested that a three-site model is more consistent with the data for the present system. At high water contents, the temperature dependence of the 7Li+ splitting vanishes, indicating counterion condensation. The behavior of the 7Li+ splitting is confirmed by measurements on DNA fibers in equilibrium with a C2H5OD-D2O-LiCl solution. The salt dependence in this system is weak. The counterion quadrupolar splitting is seen to be very sensitive to structural transitions in double-helical DNA.     

Subcellular distribution of nitroxide spin-labelled 9-aminoacridine in living KB cells

A spin-labelled derivative of 9-aminoacridine, the AATEMPO, was studied with respect to its localization in KB cells in vivo. It was found that both nuclear and mitochondrial DNAs were targets for this intercalating dye. The observed speed of intercalation and the absence of a blocked signal in cellular membranes suggested that no receptor -or carrier- proteins were implied in the penetration process. No changes in cell membrane fluidity were observed following the administration of m-AMSA, the unlabelled structural analog, to the living cells, which seemed to exclude the plasma membrane as a possible site of action of 9-aminoacridines. Side effects of phenol/chloroform mixtures and high-salt concentrations on the intercalation phenomenon were also described.     

Nature of lysozyme-water interactions by proton NMR.

Proton spin-lattice relaxation measurements were performed in 10 mM lysozyme solution as a function of temperature and degree of substitution of solvent H2O with D2O. The results show that in the temperature range from 274 to 323 K, the intermolecular lysozyme proton water proton coupling contributes appreciably to the observed water proton relaxation rate. In this system exchange between water protons and labile protein protons does not dominate the behaviour with temperature of the water-lysozyme intermolecular contribution to the spin-lattice relaxation.     

Dynamics of the phosphate group in phospholipid bilayers. A 31P angular dependent nuclear spin relaxation time study.

To understand 31P relaxation processes and hence molecular dynamics in the phospholipid multilayer it is important to measure the dependence of the 31P spin-lattice relaxation time on as many variables as the physical system allows. Such measurements of the 31P spin-lattice relaxation rate have been reported both as a function of Larmor frequency and temperature for egg phosphatidylcholine liposomes (Milburn, M.P., and K.R. Jeffrey. 1987. Biophys. J. 52:791-799). In principle, the spin-lattice relaxation rate in an anisotropic environment such as a bilayer will be a function of the angle between the bilayer normal and the magnetic field. However, the measurement of this angular dependence has not been possible because the rapid (on the time-scale of the spin-lattice relaxation rate) diffusion of the lipid molecules over the curved surface of the liposome average this dependence (Milburn, M.P., and K.R. Jeffrey. 1987. Biophys. J. 52:791-799; Brown, M.F., and J.H. Davis. 1981. Chem. Phys. Lett. 79:431-435). This paper reports the results of the measurement of the 31P spin-lattice relaxation rate as a function of this angle, beta', (the angle between the bilayer normal and the external magnetic field) using samples oriented between glass plates. These measurements were made at high field (145.7 MHz) where the spin-lattice relaxation processes are dominated by the chemical shielding interaction (Milburn, M.P., and K.R. Jeffrey. 1987. Biophys. J. 52:791-799). A model of molecular motion that includes a fast axially symmetric rotation of the phosphate group (tau i approximately 10(-9) s) and a wobble of the head group tilt with respect to this rotation axis has been used to describe both the angular dependence of the spin-lattice relaxation and the spectral anisotropy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Water and Backbone Dynamics in a Hydrated Protein

Rotational immobilization of proteins permits characterization of the internal peptide and water molecule dynamics by magnetic relaxation dispersion spectroscopy. Using different experimental approaches, we have extended measurements of the magnetic field dependence of the proton-spin-lattice-relaxation rate by one decade from 0.01 to 300 MHz for ¹H and showed that the underlying dynamics driving the protein ¹H spin-lattice relaxation is preserved over 4.5 decades in frequency. This extension is critical to understanding the role of ¹H₂O in the total proton-spin-relaxation process. The fact that the protein-proton-relaxation-dispersion profile is a power law in frequency with constant coefficient and exponent over nearly 5 decades indicates that the characteristics of the native protein structural fluctuations that cause proton nuclear spin-lattice relaxation are remarkably constant over this wide frequency and length-scale interval. Comparison of protein-proton-spin-lattice-relaxation rate constants in protein gels equilibrated with ²H₂O rather than ¹H₂O shows that water protons make an important contribution to the total spin-lattice relaxation in the middle of this frequency range for hydrated proteins because of water molecule dynamics in the time range of tens of ns. This water contribution is with the motion of relatively rare, long-lived, and perhaps buried water molecules constrained by the confinement. The presence of water molecule reorientational dynamics in the tens of ns range that are sufficient to affect the spin-lattice relaxation driven by ¹H dipole-dipole fluctuations should make the local dielectric properties in the protein frequency dependent in a regime relevant to catalytically important kinetic barriers to conformational rearrangements.     

Acridine dimers: influence of the intercalating ring and of the linking-chain nature on the equilibrium and kinetic DNA-binding parameters

The rigidity of the linking chain of bifunctional intercalators in the ditercalinium series was shown to be critical for antitumor activity. In order to study the influence of the rigidity of the linking chain on the DNA-binding properties of DNA bifunctional intercalators, fluorescent 9-aminoacridine and 2-methoxy-6-chloro-9-aminoacridine analogues with chains of variable rigidity were synthesized. 1H-NMR studies show that the conformation of 9-aminoacridine dimers is almost independent of the nature of the linking chain. A strong self-stacking of the aromatic rings of the 2-methoxy-6-chloro-9-aminoacridine is observed for dimers with flexible chains but not for those with rigid chains. All the dimers having a linking chain long enough to bisintercalate in DNA according to the excluded site model are indeed bisintercalators. The kinetic association constant of all monomers and dimers for poly[d(A-T)].poly[d(A-T)] are in the same range (2-4 x 10(7) M-1 s-1). The large increase of DNA binding affinity observed for the dimers is always associated with the expected decrease of the dissociation rate constant. The effect of chain rigidity and pH on the calf thymus DNA binding of 9-aminoacridine and 2-methoxy-6-chloro-9-aminoacridine dimers is quite different. In the series of 9-aminoacridine the pKa of the dimers remains high and therefore no difference of DNA-binding affinity is observed between pH 5 and 7.4. The rigidity of the linking chain does not significantly alter the DNA-binding affinity. In the 2-methoxy-6-chloro-9-aminoacridine series, the pKa of all dimers became smaller than the physiological pH and a dramatic decrease of DNA-binding affinity is observed when the pH is increased from pH 5 to 7.4. This decrease appears significantly smaller for dimers with rigid chains. A similar dramatic decrease of binding affinity at pH 7.4 is not observed for poly[d(A-T)].poly[d(A-T)]. This factor makes these dimers strongly specific for the alternating polymer at pH 7.4.     

Dynamics in synthetic oligonucleotides. A solid-state deuterium NMR study

Solid-state 2H NMR spectroscopy was used to investigate dynamics in the [methyl-2H]thymidine-labeled oligonucleotide [d(CGCGAAT*T*CGCG)]2. Quadrupole echo line shapes, spin-lattice relaxation, and quadrupolar echo decay rates were investigated as a function of hydration W (moles of water/moles of nucleotide) between 0 and approximately 30. The amplitude of the base motion, modeled as a fast four-site libration, or diffusion in a cone, increased slightly with higher levels of hydration. A slower component of motion about the helix axis appears at W approximately 10 and increases in rate and amplitude, leading to the intermediate rate line shape observed at W approximately 21.     

Enhancement of Mutagenic Activity of 9-Aminoacridine by Introducing a Nitro Group into the Molecule

Mutagenic activity and DNA intercalation were examined for 9-aminoacridine (9-AA) and its derivatives. Introduction of a nitro group into the 9-AA molecule was found to enhance the activity enormously as was detected by the Ames test. Acetylation of amino group at 9-position of acridine ring inhibited the intercalation, the frameshift activity disappearing. Rat liver S9 converted 9-AA metabolically to 9-amino-2-hydroxyacridine.     

Using saturation-recovery EPR to measure distances in proteins: applications to photosystem II.

The stable tyrosine radical YD. (tyrosine 160 in the D2 polypeptide) in photosystem II (PSII) exhibits nonexponential electron spin-lattice relaxation transients at low temperature. As previously reported, the tetranuclear Mn complex in PSII significantly enhances the spin-lattice relaxation of YD.. However, in Mn-depleted PSII membranes, the spin-lattice relaxation transients of YD. are also nonexponential, and progressive power saturation (P 1/2) experiments show that it does not behave like an isolated tyrosine radical. A model is developed to treat the interaction of two paramagnets in a rigid lattice at a fixed distance apart but with a random orientation in a magnetic field. This model describes the spin-lattice relaxation of a radical in proximity to another paramagnetic site in terms of three relaxation rate constants: the "intrinsic" relaxation rate, the relaxation rate due to scalar exchange, and the relaxation rate due to dipole-dipole interactions. The intrinsic and the scalar exchange relaxation rates are isotropic and together contribute a single rate constant to the spin-lattice relaxation transients. However, the dipolar relaxation rate is orientation dependent. Each orientation contributes a different dipolar relaxation rate constant to the net spin-lattice relaxation rate constant. The result is a superposition of single-exponential recoveries, each with a different net rate constant, causing the observed saturation-recovery transients to be non-(single)-exponential. Saturation-recovery relaxation transients of YD. are compared with those of a model tyrosine radical, generated by UV photolysis of L-tyrosine in a borate glass. From this comparison, we conclude that scalar exchange does not make a significant contribution to the spin-lattice relaxation of YD. in Mn-depleted PSII. We account for the nonexponential relaxation transients obtained from YD. in Mn-depleted PSII membranes in terms of dipolar-induced relaxation enhancement from the non-heme Fe(II). From simulations of the spin-lattice relaxation transients, we obtain the magnitude of the magnetic dipolar interaction between YD. and the non-heme Fe(II), which can be used to calculate the distance between them. Using data on the non-heme Fe(II) in the reaction center of Rhodobacter sphaeroides to model the non-heme Fe(II) in PSII, we calculate a YD.-Fe(II) distance of greater than or equal to 38 A in PSII. This agrees well with the distance predicted from the structure of the bacterial reaction center.     

Water of hydration in the intra- and extra-cellular environment of human erythrocytes

The proton nuclear magnetic resonance (NMR) titration method (which requires measurement of the relaxation rate at multiple measured levels of dehydration) was applied to the analysis of human erythrocytes, a hemoglobin solution, plasma, and serum. The results allowed identification of bulk water and four motionally perturbed water of hydration subfractions. Based on previous NMR studies of homopolypeptides we designated these subfractions as superbound, irrotationally bound, rotationally bound, and structured. The total water of hydration (sum of both structured and bound water subfractions) in plasma, serum, and hemoglobin ranged from 2.78 to 3.77 g H2O/g dry mass and the sum of the three bound water subfractions ranged from 1.23 to 1.72 g H2O/g dry mass. The total water of hydration on hemoglobin, as determined by (i) spin-lattice (T1) and spin-spin (T2) NMR data, (ii) quench ice-crystal imprint size, (iii) calculations based on osmotic pressure data, and (iv) two other methods, ranged from 2.26 to 3.45 g H2O/g dry mass. In contrast, the estimates of total water of hydration in the intact erythrocytes ranged from 0.34 to 1.44 g H2O/g dry mass, as determined by osmotic activity and spin-lattice titration, respectively. Studies on the magnetic-field dependence of the spin-lattice relaxation rate (1/T1 rho) of solvent water nuclei in protein solutions and in intact and disrupted erythrocytes indicated that hemoglobin aggregation exists in the intact erythrocytes and that erythrocyte disruption decreases the extent of hemoglobin aggregation. Together, the present and past data indicate that the extent of water of hydration associated with hemoglobin depends on the amount of salt present and the degree of aggregation of the hemoglobin molecules.     

Competition between Li+ and Mg2+ for ATP and ADP in aqueous solution: a multinuclear NMR study

We used 7Li NMR spin-lattice relaxation times and 31P NMR chemical shifts to study the binding of Li+ and Mg2+ to the phosphate moieties of ATP and ADP. To examine the binding of Li+ and Mg2+ to the base and ribose moieties, we used 1H and 13C NMR chemical shifts. The 7Li NMR relaxation times of Li+/Mg2+ mixtures of ATP or ADP increased with increasing concentrations of Mg2+, suggesting competition between the two ions for adenine nucleotides. No significant binding of Li+ and Mg2+ to the base and ribose moieties occurred. At the pH and ionic strength used, 2:1 and 1:1 species of the Li(+)-ATP and Li+-ADP complexes were present, with the 2:1 species predominating. In contrast, 1:1 species predominated for the Mg(2+)-ADP and Mg(2+)-ATP complexes. We calculated the Li(+)-nucleotide binding constants in the presence and absence of Mg2+ and found them to be somewhat greater in the presence of Mg2+. Although competition between Li+ and Mg2+ for ATP and ADP phosphate binding sites in solution is consistent with the 31P chemical shift data, the possibility that the Li+ and Mg2+ form mixed complexes with the phosphate groups of ATP or ADP cannot be ruled out.     

Conformation of dinucleoside phosphates: the conformation encoding hypothesis

The temperature dependence of the spin-lattice relaxation rate of nucleic bases protones and HI' of ApA, ApC, CpA and CpC (D2O, pH 7) were measured. The possible closed conformers of these dinucleoside phosphates (DNP) were computed by atom-atom potential method. On the basis of conformational calculation and experimental data the composition of closed state was determined. Besides the right-handed "canonic" conformers, the "non-canonic" right- and left-handed conformers were shown to be present in the solution of all DNP studied. It is important to note that, "canonic" conformers of DNP studied being equally probable, the possibility of the realization of "non-canonic" conformers is determined by the nucleotide sequence. It may be expected that different nucleotide sequences have unique "non-canonic" conformations. That type of dependence of the spatial organization of polynucleotides on its nucleotide sequence we call "the conformational encoding".     

Electrostatic factors in DNA intercalation

The factors that determine the binding of a chromophore between the base pairs in DNA intercalation complexes are dissected. The electrostatic potential in the intercalation plane is calculated using an accurate ab initio based distributed multipole electrostatic model for a range of intercalation sites, involving different sequences of base pairs and relative twist angles. There will be a significant electrostatic contribution to the binding energy for chromophores with a predominantly positive electrostatic potential, but this varies significantly with sequence, and somewhat with twist angle. The usefulness of these potential maps for understanding the binding of intercalators is explored by calculating the electrostatic binding energy for 9-aminoacridine, ethidium, and daunomycin in a variety of model binding sites. The electrostatic forces play a major role in the positioning of an intercalating 9-aminoacridine and a significant stabilizing role in the binding of ethidium in its sterically constrained position, but the intercalation of daunomycin is determined by the side-chain binding. Sequence preferences are likely to be determined by a complex and subtle mixture of effects, with electrostatics being just one component. The electrostatic binding energy is also unlikely to be a major determinant of the twist angle, as its variation with angle is modest for most intercalation sites. Overall, the electrostatic potential maps give guidance on how positively charged chromophores can be chemically adapted by heteroatomic substitution to optimise their binding.     

9-aminoacridine inhibits the B-Z transition of poly(dA-dT).

9-Aminoacridine is the parent compound of a family of pharmacologically active model substances that bind to DNA through intercalation between base pairs. In the present study we show that 9-aminoacridine inhibits the B-to-Z isomerization of poly(dA-dT) in conditions that otherwise cause it to occur (5 M NaCl and 123 mM Ni(ClO4)2). Higher concentrations of Ni(ClO4)2 (155 mM) are able to induce the Z-form due to the disruption of the drug-polynucleotide interaction by the metal ion. Additionally, the dye reverses the Z-form in certain conditions. Thus, the data from this study indicate that 9-aminoacridine binds preferentially to the B-form of poly(dA-dT).