Solid-state NMR and rigid body molecular dynamics to determine domain orientations of monomeric phospholamban

Solid-state NMR spectroscopy, in conjunction with rigid body molecular dynamics calculations, shows that monomeric phospholamban in lipid bilayers has two distinct helical domains, with an interhelical angle within 60-100 degrees, ruling out the possibility of a continuous alpha-helical structure for this protein.     

Weakly oriented liquid-crystal NMR solvents as a general tool to determine relative configurations

A new NMR method to determine the relative configuration of asymmetric centres is presented. It proceeds through the use of a weakly ordered solvent and the measurement of orientational order parameters. The method is illustrated by using dihydropyridone derivatives for which the orientations and the relative configurations of the asymmetric carbon atoms are determined unambiguously.     

Wideline NMR studies of oriented nylon 66

Wideline NMR techniques have been applied to both singly and doubly oriented specimens of nylon 66. Variations in the spectra obtained are observed for different orientations of the specimens relative to the applied field. These variations demonstrate that the zigzag chain axis is essentially parallel to the draw direction and that motion occurs in all of the sample. The motion is of two types: random in the mobile regions and rotation of segments about the chain axis in the rigid regions.     

1H NMR studies on dimeric 2-methylchromones

A study is presented on the configuration and conformation of 2-methylchromone-dimer, as well as on khellin-dimer, based on ¹H NMR spectroscopy.     

UV/vis, 1H, and 13C NMR spectroscopic studies to determine mangiferin pKa values

The acid constants of mangiferin (a natural xanthonoid) in aqueous solution were determined through an UV/vis spectroscopic study employing the SQUAD program as a computational tool. A NMR study complements the pK(a) values assignment and evidences a H-bridge presence on 1-C. The chemical model used was consistent with the experimental data obtained. The pK(a) values determined with this procedure were as follows: H(4)(MGF)=H(3)(MGF)(-)+H(+), pKa1 (6-H)=6.52+/-0.06; H(3)(MGF)(-)=H(2)(MGF)(2-)+H(+), pKa2 (3-H)=7.97+/-0.06; H(2)(MGF)(2-)=H(MGF)(3-)+H(+), pKa3 (7-H)=9.44+/-0.04; H(MGF)(3-)=(MGF)(4-)+H(+), pKa4 (1-H)=12.10+/-0.01; where it has been considered mangiferin C(19)H(18)O(11) as H(4)(MGF). Mangiferin UV/vis spectral behavior, stability study in aqueous solution as well as NMR spectroscopy studies: one-dimensional (1)H,(13)C, 2D correlated (1)H/(13)C performed by (g)-HSQC and (g)-HMBC methods; are also presented. pK(a) values determination of H(4)(MGF) in aqueous solution is a necessary contribution to subsequent pharmacokinetic study, and a step towards the understanding of its biological effects.     

NMR studies of bond orders in heteroaromatic systems

Fifty-seven values for the ortho-benzylic coupling constant ⁴J_{Me? C?C? H} (henceforth denoted as ⁴J_OB) were obtained for a variety of heteroaromatic systems. It was shown that a good correlation exists between ⁴J_OB when the methyl group is not α to the heteroatom and the SCF-MO bond order. This method can therefore be used as an experimental means of determining bond orders in heteroaromatic systems. An examination of bond alternation in thirteen heteroaromatic systems has given a measure of relative “degree of aromaticity” for a larger number of systems than previously reported by any single method.     

1H NMR study of partially oriented 1-phenylpropyne

The proton magnetic resonance spectrum of partially oriented 1-phenylpropyne has been studied and the values of the chemical shifts, the direct and the indirect coupling constants determined from the analysis of the spectrum. The ratios of the interproton distances have been derived. No significant deviations have been observed between the relative proton positions on the aromatic ring in 1-phenylpropyne and those expected for the regular hexagonal geometry of the benzene ring. The results are insensitive to the mode of internal rotation of the methyl group about the C? C bond.     

1H NMR of probes oriented in stretched polyethylene

¹H NMR spectra of some small molecules oriented in stretched polyethylene rods have been recorded. The band splitting due to direct dipole-dipole couplings is obtained and the corresponding orientation parameters are shown as a function of the draw ratio of the polymer and the temperature. This method is compared to that of using liquid crystals as the orienting solvent.     

Solid state NMR studies of oligourea foldamers: interaction of 15N-labelled amphiphilic helices with oriented lipid membranes

Synthetic oligomers that are derived from natural polypeptide sequences, albeit with unnatural building blocks, have attracted considerable interest in mimicking bioactive peptides and proteins. Many of those compounds adopt stable folds in aqueous environments that resemble protein structural elements. Here we have chemically prepared aliphatic oligoureas and labeled them at selected positions with (15)N for structural investigations using solid-state NMR spectroscopy. In the first step, the main tensor elements and the molecular alignment of the (15)N chemical shift tensor were analyzed. This was possible by using a two-dimensional heteronuclear chemical shift/dipolar coupling correlation experiment on a model compound that represents the chemical, and thereby also the chemical shift characteristics, of the urea bond. In the next step (15)N labeled versions of an amphipathic oligourea, that exert potent antimicrobial activities and that adopt stable helical structures in aqueous environments, were prepared. These compounds were reconstituted into oriented phospholipid bilayers and the (15)N chemical shift and (1)H-(15)N dipolar couplings of two labeled sites were determined by solid-state NMR spectroscopy. The data are indicative of an alignment of this helix parallel to the membrane surface in excellent agreement with the amphipathic character of the foldamer and consistent with previous models explaining the antimicrobial activities of α-peptides.     

2H NMR studies of glycerol dynamics in protein matrices

We use (2)H NMR spectroscopy to investigate the rotational motion of glycerol molecules in matrices provided by the connective tissue proteins elastin and collagen. Analyzing spin-lattice relaxation, line-shape properties, and stimulated-echo decays, we determine the rates and geometries of the motion as a function of temperature and composition. It is found that embedding glycerol in an elastin matrix leads to a mild slowdown of glycerol reorientation at low temperatures and glycerol concentrations, while the effect vanishes at ambient temperatures or high solvent content. Furthermore, it is observed that the nonexponential character of the rotational correlation functions is much more prominent in the elastin matrix than in the bulk liquid. Results from spin-lattice relaxation and line shape measurements indicate that, in the mixed systems, the strong nonexponentiality is in large part due to the existence of distributions of correlation times, which are broader on the long-time flank and, hence, more symmetric than in the neat system. Stimulated-echo analysis of slow glycerol dynamics reveals that, when elastin is added, the mechanism for the reorientation crosses over from small-angle jump dynamics to large-angle jump dynamics and the geometry of the motion changes from isotropic to anisotropic. The results are discussed against the background of present and previous findings for glycerol and water dynamics in various protein matrices and compared with observations for other dynamically highly asymmetric mixtures so as to ascertain in which way the viscous freezing of a fast component in the matrix of a slow component differs from the glassy slowdown in neat supercooled liquids.     

Molecular orbital studies of the NMR hydrogen bond shift

Magnetic shielding constants are calculated for the protons in XOH and XOH…OH₂ (XH, CH₃, NH₂, OH and F) molecules using a slightly extended set of atomic functions modified by gauge factors. These results are used to determine theoretical values for the NMR hydrogen bond shifts in the XOH…OH₂ systems. Such theoretical data are consistent with the few available experimental data. An analysis of the theoretical results reveals that there are three major types of shielding contribution to the NMR hydrogen bond shift; (a) a deshielding change due to the variation of the local currents on the hydrogen bonded proton; (b) a reduction in shielding from currents localized on the oxygen atom of the proton donor; (c) a deshielding contribution from currents induced on the oxygen atom of the proton acceptor. Except for the water dimer, contributions (a), (b) and (c) are of comparable importance for changes in isotropic shielding. For (H₂O)₂ contributions (a) and (c) are somewhat more important than contribution (b). Contribution (c) is almost totally responsible for the changes in the anistropies of the shielding tensors associated with the hydrogen bonded protons. The proton shielding anisotropy changes which occur on hydrogen bond formation are generally much larger than the corresponding variations in the isotropic values of the shielding tensors. This suggests that proton magnetic shielding anisotropies may be more sensitive measures of features of hydrogen bonding than are isotropic proton shielding constants.     

NMR studies of molecular mobility in uniaxially stretched oriented polymers

The effect of elasticlike uniaxial tension on molecular mobility in polymers have been studied over a wide temperature range using the broad-line NMR technique. The studies were carried out on oriented semicrystalline samples of nylon 6, poly(ethylene terephthlate), polypropylene, polyethylene, polyoxymethylene, poly(vinyl alcohol), and polytetrafluorethylene. Above the low-temperature transition the NMR spectra are reversibly transformed under tension. Increases in the second moments of the spectra are attributed to weaker molecular motion in stressed polymers. The only exception is polypropylene, in which the reverse, i.e., enhancement of molecular mobility, can be observed in a certain temperature range. In the spectra of polymers stretched above the glass transition temperature the narrow component decreases, thus indicating inhibition of micro-Brownian motion, a phenomenon we call “mechanical vitrification.” Such mechanical vitrification is proved to result from reduction in the number of possible tie-chain conformations in the non-crystalline regions and not from closer packing of chains. In discussing the results we use the experimental data on the reduction of the number of gauche isomers under tension (on average, one transition of a gauche link to the trans state causes at least five methylene groups in the main chain to become immobile). The results of studies of molecular mobility in stretched polymers are used for more accurate definition of the mechanisms of molecular motion at different temperatures. A method for evaluation of the energy of intermolecular interactions which hinder small-scale motion at low temperature is suggested.     

Wideline NMR studies of oriented nylon 66 under tensile stress

NMR linewidth studies of nylon 66 as a function of temperature and applied tensile stress have been conducted. The principal motional transition temperature was found to be shifted to higher temperatures with stress application by 9°C./g./den. At any given temperature, increased stress resulted in an increased linewidth. An attempt was made to correlate the shift in the motional transition temperature with the concept that a segment experiencing motion must do work against the applied tensile stress.     

Lateral diffusion studied by pulsed field gradient NMR on oriented lipid membranes

This mini-review focuses on the utilization of pulsed magnetic field gradients to measure diffusional motion in systems of macroscopically oriented lipid bilayers. The NMR diffusion technique is proposed to have appreciable potential for future biophysical investigations in the field of membrane biology. Topics such as transport of molecules both across and in the plane of the membrane can be successfully studied, and the formation of lipid domains and their intrinsic dynamics can also be scrutinized. First, a short introduction to the NMR technique is given together with a brief discussion on methods of obtaining a good bilayer orientation. Then, a number of recent results on biophysical/biological membrane systems of great interest is presented, in which some unique conclusions on so-called 'raft membranes' are reached. It is shown for systems with large two-phase areas of liquid disordered and liquid ordered phases that lipid lateral diffusion is faster in the former phase and has a smaller apparent activation energy. Further, on the time-scale of the experiments (50-250 ms), exchange between the two phases is fast in the phospholipid-cholesterol-water ternary system, whereas it is slow in the sphingomyelin-dioleoylphosphatidylcholine-cholesterol-water quaternary system.     

Two orientations and photochromism of polyelectrolyte/bacteriorhodopsin alternative deposition multilayers

A series of organized multilayers have been formed by the alternative adsorption of positively charged poly(dimethyldiallylammonium chloride) (PDAC) and purple membrane (PM) fragments in suspensions at pH = 4—11. Both UV-vis spectrophotometry and quartz crystal micro-balance (QCM) technique were used to monitor the deposition process of PDAC/bacteriorhodopsin (bR) multilayers, suggesting that PM fragments and PDAC are deposited alternatively on the substrate uniformly. Upon illumination, all these multilayers generate photovoltages with defined signs. The negative sign of photovoltage accompanying the formation of M-state at pH <7 indicates that the extracellular side of PM fragments is directed toward the substrate; and the positive sign at pH≥7 indicates that the cytoplasmic side of PM fragments is directed toward the substrate. In addition, the long-lived multiple M-state has been observed in all multilayer films. Moreover, M-state at high pH, which shows the longer lifetime than that at low pH, de     

Redox potential of bacteriorhodopsin in oriented purple membrane films

The redox potentials of bacteriorhodopsin (BR) in oriented purple membrane (PM) films formed by the electrophoretic sedimentation (EPS) technique and the Langmuir–Blodgett (LB) technique were measured. The PM LB film and PM EPS film formed at 0.4 V versus SCE electrode potential exhibited two redox potentials at −0.48 and −0.76 V for the cis- and trans-isomers of the retinal Schiff base in the BR, respectively. The reduction current of BR in the PM LB film was greater than that in the PM EPS film and the reduction current of BR in the PM cast film was ca. 70% smaller values of the PM LB film. The results indicate that the electron transfer was affected by a difference in the orientation of PM patches in the films.     

Dynamic 1H NMR study around the carbon-carbon single bond and partial carbon-carbon double bond in the two particular phosphorus ylides and in an enaminoester

Herein, a series of separate dynamic (1)H NMR effects are reported at different temperatures within a particular enaminoester involving a phenanthridine. These effects are attributed to restricted rotation around the two single bonds such as carbon-carbon (H(a)-C-C-H(b)) and nitrogen-carbon (NCCOOCH(3)). Activation energies (E(a)) for these interconversion processes in their rotational isomers are equal to 20 and 20 ± 1 kJ mol(-1), respectively. In addition, three dynamic (1)H NMR effects are investigated at different temperatures for a particular phosphorus ylide involving a 2-indolinone around the carbon-carbon single bond (H-C-C-PPh(3)) within the two Z- and E-rotational isomers and partial carbon-carbon double bond (OCH(3)-C=C-PPh(3)). Activation energies (E(a)) for these interconversion processes in rotational isomers are equal to 53, 63 and 73 ± 1 kJ mol(-1) , respectively. This behavior was also observed for other phosphorus ylide containing 2-mercaptobenzoxazole around the carbon-carbon single bond and partial carbon-carbon double bond with their relevant activation energies containing 13, 10 and 75 ± 1 kJ mol(-1), respectively.     

1H NMR studies on thioamides: Barriers to internal rotation

The barrier to the internal rotation of the dimethylamino group in thioamides of structure R? CS? N(CH₃)₂, R being (CH₃)₂,N? CS? , CH₃O₂C? or N?C? , is studied by proton magnetic resonance, using the lineshape analysis method of Nakagawa. In the solvents o-dichlorobenzene, naphthalene and nitrobenzene all ΔG≠ values are in the range of 23 to 24 kcal/mol. In these solvents the E_a and ΔS≠ values of each product are linearly related to the dielectric constants.