To investigate cytosine protonation and its influence on the sequence-dependent thermal stability of DNA triplexes in detail, we have employed homo- and heteronuclear NMR experiments on specifically (15)N-labeled oligodeoxynucleotides that were designed to fold into intramolecular triple helices of the pyrimidine motif under appropriate conditions. These experiments reveal that cytosines in central positions of the triplex are significantly protonated even at neutral pH. However, semiprotonation points for individual cytosine bases as determined from pH-dependent measurements show considerable differences depending on their position. Thus, protonation is disfavored for adjacent cytosines or for cytosines at the triplex termini, resulting in a smaller contribution to the overall free energy of the triple helical system. In contrast, protonation of the base upon substitution of 5-methylcytosine for cytosine in the triplex third strand is only affected to a minor extent, and triplex stabilization by the methyl substituent is shown to primarily arise from stacking energies and/or hydrophobic effects.

The anisotropy and pulse frequency dispersion of the spin–spin relaxation time TCP2E from Carr–Purcell–Meiboom–Gill pulse sequences is employed to evaluate the major contribution to transverse 2H spin relaxation in bilayer membranes. Analysis of the experiments is achieved in terms of a density operator formalism, employing the stochastic Liouville approach. From a comparison of the observed angular and frequency dependences of TCP2E with those predicted for order director fluctuations, we conclude that collective lipid motions constitute the dominant transverse relaxation process. Computer simulations provide the viscoelastic parameters of the lipid membranes. For 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayers at T=318 K an average elastic constant of K=2×10−11 N and an effective viscosity of η=0.1 P have been determined. Using the experimentally accessible value for the long wavelength cutoff of the elastic modes, one obtains the mean square amplitude of the director fluctuations 〈θ20〉=0.04. This corresponds to an order parameter of SOF=0.94. Apparently, the contributions of the collective motions to the measured order parameters are marginal.

The influence of cholesterol on the dynamic organization of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers was studied by deuteron nuclear magnetic resonance (2H NMR) using unoriented and macroscopically aligned samples. Analysis of the various temperature- and orientation-dependent experiments were performed using a comprehensive NMR model based on the stochastic Liouville equation. Computer simulations of the relaxation data obtained from phospholipids deuterated at the 6-, 13- and 14-position of the sn-2 chain and cholesterol labeled at the 3 alpha-position of the rigid steroid ring system allowed the unambiguous assignment of the various motional modes and types of molecular order present in the system. Above the phospholipid gel-to-liquid-crystalline phase transition, TM, 40 mol % cholesterol was found to significantly increase the orientational and conformational order of the phospholipid with substantially increased trans populations even at the terminal sn-2 acyl chain segments. Lowering the temperature continuously increases both inter- and intramolecular ordering, yet indicates less ordered chains than found for the pure phospholipid in its paracrystalline gel phase. Trans-gauche isomerization rates on all phospholipid alkyl chain segments are slowed down by incorporated cholesterol to values characteristic of gel-state lipid. However, intermolecular dynamics remain fast on the NMR time scale up to 30 K below TM, with rotational correlation times tau R parallel for DMPC ranging from 10 to 100 ns and an activation energy of ER = 35 kJ/mol. Below 273 K a continuous noncooperative condensation of both phospholipid and cholesterol is observed in the mixed membranes, and at about 253 K only a motionally restricted component is left, exhibiting slow fluctuations with correlation times of tau R perpendicular greater than 1 microsecond. In the high-temperature region (T greater than TM), order director fluctuations are found to constitute the dominant transverse relaxation process. Analysis of these collective lipid motions provides the viscoelastic parameters of the membranes. The results (T = 318 K) show that cholesterol significantly reduces the density of the cooperative motions by increasing the average elastic constant of the membrane from K = 1 x 10(-11) N for the pure phospholipid bilayers to K = 3.5 x 10(-11) N for the mixed system.

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The solution structure of the DNA decamer d(CATTTGCATC)-d(GATGCAAATG), comprising the octamer motif of immunoglobulin genes, is determined by restrained molecular dynamics (rMD) simulations. The restraint data set includes interproton distances and torsion angles for the deoxyribose sugar ring which were previously obtained by a complete relaxation matrix analysis of the two-dimensional nuclear Overhauser enhancement (2D NOE) intensities and by the quantitative simulation of cross-peaks in double-quantum-filtered correlated (2QF-COSY) spectra. The influence of torsion angles and the number of experimental distance restraints on the structural refinement has been systematically examined. Omitting part of the experimental NOE-derived distances results in reduced restraint violations and lower R factors but impairs structural convergence in the rMD refinement. Eight separate restrained molecular dynamics simulations were carried out for 20 ps each, starting from either energy-minimized A- or B-DNA. Mutual atomic root-mean-square (rms) differences among the refined structures are well below 1 A and comparable to the rms fluctuations of the atoms about their average position, indicating convergence to essentially identical structures. The average refined structure was subjected to an additional 100 ps of rMD simulations and analyzed in terms of average torsion angles and helical parameters. The B-type duplex exhibits clear sequence-dependent variations in its geometry with a narrow minor groove at the T3.A3 tract and a large positive roll at the subsequent TG.CA step. This is accompanied by a noticeable bend of the global helix axis into the major groove. There is also evidence of significant flexibility of the sugar-phosphate backbone with rapid interconversion among different conformers.