31P nmr spin-lattice relaxation studies of deoxyoligonucleotides

Temperature-dependent conformational transitions of deoxyoligonucleotides have been monitored by measuring ³¹P chemical shifts, spin-lattice relaxation times (T₁), and ³¹P-{H} nuclear Overhauser enhancements (NOEs). The measured NOE ranged from 30 to 80%, compared to the theoretical maximum of 124% for a dipolar relaxation mediated by rapid isotropic rotation. The observed 3′-5′ phosphate diester ³¹P T₁ showed a similar temperature dependence over the range 2–75°C for both double- and single-stranded oligonucleotides, and for dinucleotides. The results show that dipole–dipole interactions dominate the internucleotide phosphate relaxation rate in oligonucleotides. The same is true of terminal phosphate groups at low temperature; but at higher temperature another process, possibly due to contamination by paramagnetic ions, becomes dominant. The rotational correlation time τ_R calculated from the dipole–dipole relaxation rate of the internucleotide phosphate in d(pA)₂ at 16°C is τ_R = 5.0 × 10^?10 sec, implying a Stokes radius for isotropic rotation of 7.6 Å. The T₁ and NOE values for the double-helical octanucleotide d(pA)₃pGpC(pT)₃ are consistent with dominance of dipole–dipole relaxation and isotropic rotation of a sphere of radius 14 Å, a reasonable dimension for the double helix. Activation energies for the rotation of dinucleotides range from 4 to 6 kcal/mol, close to the value of 4 kcal/mol expected for isotropic rotation. In order to test the possible effect of internal motion of correlation time τ_G on the results, we considered a model in which the nucleotide chain rotates about the P-O bonds. Comparison of the calculation with our experimental results shows that internal motion with τ_G ? 10^?9 sec, as found from other studies to be present for large nucleic acids, would not influence out T₁ and NOE values enough to be distinguished from isotropic rotation. However, we can conclude that τ_G cannot be as fast as 10^?10 sec, even for dinucleotides.     

Proton and phosphorus magnetic relaxation studies on the dynamic structure of single-stranded polyriboadenylic acid

Proton and phosphorus-31 nuclear spin-lattice relaxation times (T₁) have been measured with the Fourier-transform method at 100 and 40.5 MHz, respectively, on single-stranded polyriboadenylic acid (poly(A)) in a neutral D₂O solution in the temperature range of 14–82°C. T₁ minimum is observed around 35–45°C for H(8), H(1′), and phosphorus resonances. Rotational correlation times have been deduced from the T₁ data, which indicate that the sugar–phosphate backbone as well as the base–sugar segment is undergoing rapid internal motion of 10^?8–10^?10 sec range. The molecular motion of the sugar–phosphate backbone as deduced from the phosphorus relaxation is well-characterized by a single activation enthalpy of 8.1 kal/mole for the whole temperature range of 14–82°C. Activation enthalpies of similar magnitude have been obtained for the motion of the adenine–ribose moiety from H(8) and H(1′) relaxation. The relative magnitude of T₁ for H(8) and H(1′) infers that the poly(A) nucleotide exists on the average as anti in the single-stranded form. The phosphorus T₁ value is consistent with a conformation such that both C(4′)–C(5′) and C(4′)–C(3′) bonds are nearly trans to their connected O–P bonds.     

Interaction of the antimalarial drug fluoroquine with DNA,tRNA, and poly(A): 19F-NMR chemical-shift and relaxation,optical absorption,and fluorescence studies

The interaction of the fluorinated antimalarial drug fluoroquine [7-fluoro-4-(diethyl-amino-1-methylbutylamino)quinoline] with DNA, tRNA, and poly(A) has been investigated by optical absorption, fluorescence, and ¹⁹F-nmr chemical-shift and relaxation methods. Optical absorption and fluorescence experiments indicate that fluoroquine binds to nucleic acids in a similar manner to that of its well-known analog chloroquine. At low drug-to-base pair ratios, binding of both drugs appears to be random. Fluoroquine and chloroquine also elevate the melting temperature (T_m) of DNA to a comparable extent. Binding of fluoroquine to DNA, tRNA, or poly(A) results in a downfield shift of about 1.5 ppm for the ¹⁹F-nmr resonance. The chemical shift of free fluoroquine depends on the isotopic composition of the solvent (D₂O vs H₂O). The solvent isotope shift is virtually eliminated by fluoroquine binding to any one of the nucleic acids. ¹⁹F-nmr relaxation experiments were carried out to measure the spin-lattice relaxation time (T₁), ¹⁹F{¹H} nuclear Overhauser effect (NOE), off-resonance intensity ratio (R), off-resonance rotating-frame spin-lattice relaxation time (T), and linewidth for fluoroquine in the nucleic acid complexes. By accounting for intramolecular proton-fluorine dipolar and chemical-shift anisotropy contributions to the fluorine relaxation, all of the relaxation parameters for the fluoroquine–DNA complex can be well described by a motional model incorporating long-range DNA bending on the order of a microsecond and an internal motion of the drug on the order of a nanosecond. Selective NOE experiments indicate that the fluorine in the drug is near the ribose protons in the RNA complexes, but not in the DNA complex. Details of the binding evidently differ for the two types of nucleic acids. This study provides the foundation for an investigation of fluoroquine in intact cells.     

31P-NMR studies of DNA in nucleosome core particles

³¹P Nmr parameters (δ, T₁, W_1/2, and NOE) were measured for the DNA in nucleosome core particles at three frequencies and compared with similar data for the histone-free DNA. An essentially linear relationship was found between the frequency of observation and line-width for the single broad envelope of ³¹P resonances of the DNA in the nucleosome cores. We attributed this largely to chemical shift dispersion, with smaller contributions from chemical shift anisotropy and dipolar broadening. These results suggest the presence of different environments for phosphorus atoms in the core particles. However, within the accuracy of the method, no asymmetry in the resonance could be detected, which would tend to rule out any significant degree of DNA “kinking.” To investigate the interactions of the DNA and histones within the core particles we also studied transitions induced by urea and by temperature. Urea caused two stepwise increases in linewidth, which we attributed to conformational changes. A biphasic transition was also observed in the temperature profile, consistent with previous optical studies [Weischet et. al. (1978) Nucleic Acids Res. 5 , 139]. Various models with different types of local mobility were examined by the relaxation theory. A model of isotropic motion having a broad distribution of correlation times gave a fairly good fit to the ³¹P-nmr data.     

Dielectric relaxation of DNA solutions. IV. Effects of salts and dyes

The effects of salts (NaCl, LiCl, Me₄NCl, AgNO₃, MgCl₂, CuCl₂ and MnCl₂) and dyes (acridine orange and methylene blue) on the low-frequency dielectric relaxation (0.1 Hz–30 kHz) of dilute aqueous solutions of DNA were investigated with varying salt or dye concentrations. Both the dielectric relaxation time τ_D and the rotational relaxation time τ estimated from the reduced viscosity decrease in quite parallel ways with increasing M/P (M/P being the normality ratio of cation to phosphate residue), reflecting the contraction of DNA molecule due to electrostatic shielding and cation binding. The agreement between τ_D and τ through the whole range of M/P supports our previous conclusion that the low-frequency relaxation of DNA arises from rotation of the molecule. The dielectric increment Δε also decreases with increasing M/P on account of both the contraction of DNA and the decrease in effective degree of dissociation of DNA. Δε as a function of M/P is interpreted in terms of a quasi-permanent dipole due to counterion fluctuation. These effects of cations are the strongest for divalent cations and rather weak for Na⁺, Li⁺, and Me₄N⁺. Effects of dye on τ_D and Δε are also well explained by the rotation of DNA molecule with a quasi-permanent dipole due to counterion fluctuation on the basis of intercalation of dye at D/P < 0.2 (D/P being the molarity ratio of dye to phosphate residue) and external binding at 0.2 < D/P < 1.0.     

1H-nmr comparative studies of polynucleotides: Conformation and dynamic structure of polyribo(uridylic) and polyribo(cytidylic) acids in neutral solution

The conformation and the dynamic structure of single-stranded poly(U) and poly(C) in neutral aqueous solution have been investigated by ¹H-nmr at two different frequencies (90 and 250 MHz) and at various temperatures. Measurements of proton chemical shifts, coupling constants J_H-H, and proton relaxation times, T₁, T₂, versus temperature show a striking difference in conformation and in dynamic structure between the two polynucleotides studied. The temperature effect on δ and J_H-H is found to be substantial for poly(C) and insignificant for poly(U). The S conformer is favored in poly(U), whereas the N conformer strongly predominates in poly(C) (?90%), similar to the case for RNAs. These results suggest that single-stranded poly(C) probably possesses a helical or partial helical structure, whereas poly(U) shows a clear preference for the random coil, in agreement with the optical results. The local motions of the ribose and base were studied at various temperatures by measurements on the relaxation times at 90 and 250 MHz. For a given temperature between 22 and 72°C, the ratio T₁(90)/T₁(250) is practically the same for all poly(U) protons, indicating that in this temperature interval the ribose base unit of poly(U) undergoes an isotropic motion characterized by a single correlation time τ_c. Above 52°C, poly(C) exhibits a dynamic structure similar to poly(U). Below this temperature, poly(C) exists in an equilibrium between randomly coiled and single-stranded helix forms. This situation is characterized by a strong cross-relaxation effect and T₁ values corresponding to a relatively short apparent correlation time. An activation energy of 4 kcal/mol was determined for the motion of the ribose–base unit in both single-stranded polynucleotides.     

Frequency dependence of the proton magnetic relaxation in aqueous solutions iof haemoproteins

The longitudinal proton magnetic relaxation times T₁ were measured for ferri (met)-and carbonmonoxy-bovine haemoglobin and equine myoglobin in 0.1 M KH₂PO₄ aqueous solutions near pH 6 at 5°C and 35°C from 1.5- to 60-MHz Larmor frequencies. It is concluded that the correlation time τ_C for the dipole–dipole interaction of electron and nuclear spins is in fact the electron (ferric) spin relaxation time τ_S being close to 1.5 × 10^?10 sec for both metHb and metMb at 5°C. At 35°C the paramagnetic relaxation rates are not determined solely by the relaxation of protons exchanging from the haem pocket with bulk solvent. Hence, τC at 35°C cannot be calculated from the dispersion data obtained at this temperature. The relevance of this for the determination of interspin distances r is discussed.     

Histone-induced conformational changes in DNA as probed by quasi-elastic light scattering.

Quasi-elastic light scattering as measured by intensity fluctuation (self-beat) spectroscopy in the time domain can be profitably used to follow both the translational diffusion D and the dominant internal flexing mode τ_int of DNA and its complexes with various histones in aqueous salt solutions. Without histones, DNA is found to have D = 1.6 × 10^?8 cm²/sec and τ_int ? 5 × 10^?4 sec in 0.8 M NaCl, 2 M urea at 20°C. Total histone as well as fraction F2A induce supercoiling (D = 2.6 × 10^?8 cm²/sec, τ_int ? 2.8 × 10^?4 sec) whereas fraction F1 induces uncoiling (D = 1.0 × 10^?8 cm²/sec, τ_int ? 9.4 × 10^?4 sec). Upon increasing the salt concentration to 1.5 M the DNA–histone complex dissociates (D = 1.8 × 10^?8 cm²/sec). Upon decreasing the salt concentration to far below 0.8 M, the DNA–histone complex eventually precipitates as a chromatin gel.     

31P NMR studies of the solution structure and dynamics of nucleosomes and DNA.

31P NMR studies of 140 base pair DNA fragments in nucleosomes and free in solution show no detectable change in the internucleotide 31P chemical shift or linewidth when DNA is packaged into nucleosomes. Measurements of 31P spin-lattice relaxation times T1 and 31P-[H] nuclear Overhauser enhancements revealed internal motion with a correlation time of about 4 x 10(-10) sec in double helical DNA, both free in solution and bound to nucleosomal core proteins. This result implies greater dynamic mobility in double helical DNA than has previously been supposed.     

Dielectric relaxation of collagen in aqueous solutions

The complex dielectric constant of collagen in aqueous solutions (polymer concentration, C_p = 0.02–0.2%) was measured at 10°C in the frequency range from 3 Hz to 30 kHz. The loss peak for C_p = 0.02% is located at 90 Hz and the dielectric relaxation time τ_D is estimated to be 1.8 ± 0.3 msec. The τ_D agrees well with the rotational relaxation time estimated from the reduced viscosity, and the relaxation is ascribed to the end-over-end rotation of the molecule. The C_p dependence of τ_D and the dielectric increment Δε are interpreted in terms of the aggregation of molecules. The dipole moment of a molecule, obtained from Δε at C_p = 0.02% and pH 6.5, is (5.2 ± 0.2) × 10⁴D, which is explained by the asymmetrical distribution of the ionized side chains of the molecule.     

Dielectric relaxation of DNA solutions. III. Effects of DNA concentration, protein contamination, and mixed solvents

As a continuation of previous papers [Biopolymers (1976) 15 , 879; (1978) 17 , 1508], the low-frequency dielectric relaxation of DNA solutions was studied with a four-electrode cell and the simultaneous two-frequency measurement. Below a critical concentration, the dielectric relaxation time agrees with the rotational relaxation time estimated from the reduced viscosity and is almost independent of DNA concentration C_p, and the dielectric increment is proportional to C_p. The critical concentration is approximately 0.02% of DNA for molecular weight M_r 2 × 10⁶ and 0.2% for M_r 4.5 × 10⁵ in 1 mM NaCl. Dielectric relaxations are compared for samples before and after deproteinization, and the protein contamination is found to have a minor effect on the dipole moment of DNA. The effect of a mixed solvent of water and ethanol on the dielectric relaxation of DNA is well interpreted in terms of changes in viscosity and the dielectric constant of the solvent, assuming that the relaxation arises from rotation of the molecule with a quasi-permanent dipole due to counterion fluctuation.     

Dielectric relaxation of DNA solutions. II

Real and imaganiry parts of complex dielectric constant of dilute solutions of DNA in 10^?3M NaCl with molecular weight ranging from 0.4 × 10⁶ to 4 × 10⁶ were measured at frequencies from 0.2 Hz to 30 kHz. Dielectric increments Δε were obtained from Cole-Cole plots and relaxation times τ_D from the loss maximum frequency. The τ_D of all samples agrees well with twice of the maximum viscoelastic relexation time in the Zimm theory, indicating that the low-frequency dielectric relaxiation should be ascribed to be the rotation of DNA. The rms dipole moment, which was obtained from Δε, agree well with that calculated from the counterion fluctuation theory. The dielectric increment was found to be greatly depressed in MgCl₂, which is resonably interpreted in terms of a strong binding of Mg⁺⁺ ions with DNA.     

Conformation and dynamic structure of poly(inosinic acid) in neutral aqueous solution by 1H-, 2H-, 13C-, and 31P-NMR spectroscopy

The conformation and dynamic structure of single-stranded poly(inosinic acid), poly(I), in aqueous solution at neutral pH have been investigated by nmr of four nuclei at different frequencies: ¹H (90 and 250 MHz), ²H (13.8 MHz), ¹³C (75.4 MHz), and ³¹P (36.4 and 111.6 MHz). Measurements of the proton-proton coupling constants and of the ¹H and ¹³C chemical shifts versus temperature show that the ribose is flexible and that base-base stacking is not very significant for concentrations varying from 0.04 to 0.10M in the monomer unit. On the other hand, the proton T₁ ratios between the sugar protons, T₁ (H₁′)/T₁ (H₃′), indicate a predominance of the anti orientation of the base around the glycosidic bond. The local motions of the ribose and the base were studied at different temperatures by measurements of nuclear Overhauser enhancement (NOE) of protonated carbons, the ratio of the proton relaxation times measured at two frequencies (90 and 250 MHz), and the deuterium quadrupolar transverse relaxation time T₂. For a given temperature between 22 and 62°C, the ¹³C-{¹H} NOE value is practically the same for seven protonated carbons (C₂, C₈, C_1′, C_2′, C_3′, C_4′, C_5′). This is also true for the T₁ ratio of the corresponding protons. Thus, the motion of the ribose–base unit can be considered as isotropic and characterized by a single correlation time, τ_c, for all protons and carbons. The τ_c values determined from either the ¹³C-{¹H} NOE or proton T₁ ratios, T₁(90 MHz)/T₁(250 MHz), and/or deuterium transverse relaxation time T₂ agree well. The molecular motion of the sugar-phosphate backbone (O-P-O) and the chemical-shift anisotropy (CSA) were deduced from T₁ (³¹P) and ³¹P-{¹H} NOE measurements at two frequencies. The CSA contribution to the phosphorus relaxation is about 12% at 36.4 MHz and 72% at 111.6 MHz, corresponding to a value of 118 ppm for the CSA (σ = σ∥ ? σ?). Activation energies of 2–6 kcal/mol for the motion of the ribose–base unit and the sugarphosphate backbone were evaluated from the proton and phosphorus relaxation data.     

Molecular mobility of polypeptides containing proline as determined by 13C magnetic resonance

The molecular conformations and dynamics of poly(L -prolyl), poly(hydroxyl-L -prolyl), poly(L -prolyl-glycyl), poly(hydroxyl-L -prolyl), and poly(glycyl-glycyl-L -prolyl-glycyl), in aqueous solution, have been studied using ¹³C pulse Fourier transform nmr spectroscopy. From a measurement of the intensities of major and minor resonances in the spectra of the copolypeptides, it was determined that 15–20% of the glycyl-prolyl and glycyl-hydroxyprolyl peptide bonds are cis. Effective rotational correlation times (τ_eff), obtained from measurements of spin-lattice relaxation times (T₁) of individual backbone and side-chain carbons, demonstrated that backbone reorientation is approximately isotropic for the five polypeptides and is characterized by correlation times of ca. 0.3–0.6 nanoseconds as a result of rapid segmental motion. In a given polypeptide glycyl and pyrrolidine residues were found to have the same backbone correlation times, but backbone carbon τ_eff values did decrease as the glycyl content of the peptides increased. A semi-quantitative analysis of C^β, C^γ, and C^δ correlation times suggests that rapid ring motion in both prolyl and hydroxyprolyl involves primarily C^γ and C^β, with the prolyl ring being more mobile than the hydroxyprolyl ring.     

Anisotropic rotational motions of poly(L-glutamic acid) in the alpha-helix conformation

The anisotropic rotational motion of the backbone and the side chains of poly(L -glutamic acid) in the α-helical structure was investigated using the ¹³C-T₁ and T₂ relaxation times of all carbon atoms with directly attached protons, obtained at a ¹³C-Larmor frequency of 67.89 MHz. The evaluation of the nmr data was carried out according to the previously derived anisotropic diffusion model, in which the macromolecule is considered a rigid rod. The rotation of the backbone is characterized by two diffusion constants, D₁ and D₃, describing the rotation perpendicular to and around the symmetry axis. The additional internal motion of the C^β-methylene group is described as a jump process with a jump rate, k₁, between two allowed rotametric states. Steric considerations indicate that the occupation of the third rotameric position is forbidden. The rotation of the C^γ-methylene group is decribed as a one-dimensional diffusion process around the C^β–C^γ bond. Investigation of the temperature dependence of the relaxation parameters led to the temperature dependence of the dynamic parameters. Activation energies were determined from these data. The dynamic parameters obtained for poly(L -glutamic acid) at 291 K are compared with the corresponding results of a previous study of poly(L -lysine). The development of an anisotropic diffusion model for the motions of the rod-shaped poly(L -lysine) α-helix and its application to the interpretation of the ¹³C-relaxation data of this molecule have already been published previously. In this model, both the overall molecular tumbling and the various internal motions have been characterized by diffusion constants or jump rates typical for each process. These dynamic parameters can be calculated from the spin–lattice relaxation times, the spin–spin relaxation times and the NOE factors of the C^α, C^β, and C^γ nuclei of the polypetide. In the present paper, we describe the application of the above-mentioned dynamic model to the interpretation of ¹³C-relaxation studies of a further homopolypeptide, poly(L -glutamic acid), in the α-helical structure. Furthermore, we studied the temperature dependence of the relaxation times of this polymer and determined the anisotropic diffusion parameters at each temperature. From their temperature dependence and from comparison of our present results with the data of our previous study of poly(L -lysine), we were able to derive new insights into the intramolecular diffusion processes and the excitation of various motions.     

A pulsed N.M.R study of D2O bound to 1,2 dipalmitoyl phosphatidylcholine

Spin lattice relaxation times in both the lab and rotating frame, have been measured for deuterons (²H) in a number of unsonicated dispersions of 1,2 dipalmitoyl phosphatidylcholine in D₂O over a range of resonant frequencies from 13 MHz to 1 MHz for temperatures from ?20°C to 65°C.The proton (¹H) spin lattice relaxation time for the lecithin was measured for resonant frequencies of 8.5 MHz, and 40 MHz over a similar range of temperatures.The results agree with broadline measurements by Salsbury et al. [1], and for the liquid crystal phase are consistent with an anisotropic tumbling model of the water molecules bound to the lecithin headgroup. This tumbling occurs with correlation times of ≤10^?10 sec and ≈ 10^?6 sec about axes parallel to and perpendicular to the bisector of the D-O-D angle within a D₂O molecule, hydrogen bonded to the negatively charged phosphate headgroup.     

Proton and phosphorus-31 magnetic relaxation studies on the interaction of polyriboadenylic acid with Mn2+

Proton and phosphorus-31 nuclear spin–lattice relaxation times T₁ and spin–spin relaxation times T₂ have been measured on the single-stranded polyriboadenylic acid [poly(A)]–Mn²⁺ system in a neutral D₂O solution in the temperature range 10°–90°C at 100 and 40.5 MHz, respectively, with the Fourier transform nmr method. Minimum values of T₁ have been found for all these nuclei, which have enabled the exact estimation of apparent distances from Mn²⁺ to H₂, H₈, H_1′, and the phosphorus nucleus to be 4.7, 4.1, 5.2, and 3.0 Å, respectively. The electron spin of Mn²⁺ penetrates into the phosphorus nucleus, giving ³¹P hyperfine coupling of more than 10⁶ Hz. Evidence of penetration of the electron spin into H₈ and H₂ is also obtained, suggesting direct coordination of nitrogen atoms of the adenine ring to the Mn²⁺ Ion. Combined with the result from proton relaxation enhancement of water, it is concluded that every Mn²⁺ ion added is bound directly to two phosphate groups with a Mn²⁺–phosphorus distance of 3.3 Å, while a part of the Mn²⁺ ions are simultaneouly bound to the adenine ring. It is estimated that 39 ± 13% and 13 ± 5% of Mn²⁺ are coordinated by N₇ and N₃ (or N₁), respectively. The motional freedom of poly(A) in the environment of the Mn²⁺ binding site has been found to be quenched to the extent that the rotational motion becomes several times slower than that of the corresponding Mn²⁺–free poly(A). The activation energies for the molecular motion are, however, practically unchanged from those for Mn²⁺–free poly(A), and are found to be 8.3, 8.5, 6.1, and 8.7 kcal/mol for H₈, H₂, H_1′, and phosphorus, respectively. T₂ of phosphorus is determined by the dissociation rate (k_?1) of Mn²⁺ from the phosphate group for the whole temperature range studied with activation enthalpy of 6.5 kcal/mol. The dissociation rates of Mn²⁺ from the adenine ring are also estimated from proton T₂ values below 50°C.     

Intensity fluctuation spectroscopy and transfer RNA conformation. III. Influence of NaCl concentration on the size and shape of the initially salt-free tRNA in solution.

The influence of sodium ion concentration in solution on the initially salt-free conformation of bulk tRNA from baker's yeast has been investigated by means of photon correlation spectroscopy. From the measured values of translational (D^T) and rotational (D^R) diffusion coefficients, the semiaxes of an ellipsoid of revolution, which are hydrodynamically equivalent to the tRNA molecule, were calculated for tRNA solutions in pure H₂O as well as in 0.005, 0.1, 0.5M NaCl and 0.01M MgCl₂ solutions at pH 4.2 and 7.5. These data, combined with our previous studies, suggested a model which describes the formation of an ordered tRNA structure due to increasing NaCl concentrations. Furthermore, we have obtained information concerning intermolecular interactions between tRNA molecules in solution. In low-salt or salt-free tRNA solutions, we detected in the linewidth distribution function an extra-fast component which can be attributed as possibly due to charge fluctuations related to the reaction of ionization of organic bases. In our light-scattering linewidth measurements, we do not see fluctuations of charged and uncharged states directly as concentration fluctuations. Rather, we postulate a modulation of long-range intermolecular electrostatic interactions between the tRNA molecules due to such charge fluctuations. It is this modulation which is related to the fast component of the time correlation function at finite concentrations. A quantitative theory is needed to provide a more definitive explanation of the dynamical behavior of tRNA in salt-free or low-salt solutions.     

An investigation of the dynamics of spermine bound to duplex and quadruplex DNA by <Superscript>13</Superscript>C NMR spectroscopy

A detailed analysis of the ¹³C relaxation of ¹³C-labelled spermine bound to duplex and quadruplex DNA is presented. T₁, T₂ and heteronuclear NOE data were collected at four ¹³C frequencies (75.4, 125.7, 150.9 and 201.2 MHz). The data were analyzed in terms of a frequency-dependent order parameter, S ²(ω), to estimate the generalized order parameter and the contributions to the relaxation from different motional frequencies in the picosecond–nanosecond timescale and from any exchange processes that may be occurring on the microsecond–millisecond timescale. The relaxation data was surprisingly similar for spermine bound to two different duplexes and a linear parallel quadruplex. Analysis of the relaxation data from these complexes confirmed the conclusions of previous studies that the dominant motion of spermine is independent of the macroscopic tumbling of the DNA and has an effective correlation time of ∼50 ps. In contrast, spermine bound to a folded antiparallel quadruplex had faster relaxation rates, especially R ₂. As with the other complexes, a fast internal motion of the order of 50 ps makes a substantial contribution to the relaxation. The generalized order parameter for spermine bound to duplex DNA and the linear quadruplex is small but is larger for spermine bound to the folded quadruplex. In the latter case, there is evidence for exchange between at least two populations of spermine occurring on the microsecond–millisecond timescale. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Proton relaxation studies in H 2 O-D 2 O mixtures. The binding of manganese (II) to bovine serum albumin

We have used the direct method for determining longitudinal relaxation times of water protons in H₂OD₂O mixtures. The relaxation time of pure water (2.7 sec) increases to 9.0 sec in 10% H₂O-90% D₂O mixture. This larger relaxation time enables us to use the direct method to accurately determine relaxation rate enhancements due to paramagnetic metal ions. The binding parameters for the interaction of manganese(II) to bovine serum albumin determined by this method are in excellent agreement with those determined earlier using pulsed methods.