Swollen liquid-crystalline lamellar phase based on extended solid-like sheets.

Ordering particles at the nanometre length scale is a challenging and active research area in materials science. Several approaches have so far been developed, ranging from the manipulation of individual particles to the exploitation of self-assembly in colloids. Nanometre-scale ordering is well known to appear spontaneously when anisotropic organic moieties form liquid-crystalline phases; this behaviour is also observed for anisotropic mineral nanoparticles resulting in the formation of nematic, smectic and hexagonal mesophases. Here we describe a lyotropic liquid-crystalline lamellar phase comprising an aqueous dispersion of planar solid-like sheets in which all the atoms involved in a layer are covalently bonded. The spacing of these phosphatoantimonate single layers can be increased 100-fold, resulting in one-dimensional structures whose periodicity can be tuned from 1.5 to 225 nanometres. These highly organized materials can be mechanically or magnetically aligned over large pH and temperature ranges, and this property can be used to measure residual dipolar couplings for the structure determination of biomolecules by liquid-state NMR. We also expect that our approach will result in the discovery of other classes of mineral lyotropic lamellar phases.

Magnetization transfer from laser-polarized xenon to protons located in the hydrophobic cavity of the wheat nonspecific lipid transfer protein.

Nonspecific lipid transfer protein from wheat is studied by liquid-state NMR in the presence of xenon. The gas-protein interaction is indicated by the dependence of the protein proton chemical shifts on the xenon pressure and formally confirmed by the first observation of magnetization transfer from laser-polarized xenon to the protein protons. Twenty-six heteronuclear nOes have allowed the characterization of four interaction sites inside the wheat ns-LTP cavity. Their locations are in agreement with the variations of the chemical shifts under xenon pressure and with solvation simulations. The richness of the information obtained by the noble gas with a nuclear polarization multiplied by approximately 12,000 makes this approach based on dipolar cross-relaxation with laser-polarized xenon promising for probing protein hydrophobic pockets at ambient pressure.

Synthesis and NMR study of a heptasaccharide, epitope of the stage-specific embryonic antigen-1 (SSEA-1).

This paper describes an efficient synthesis of the beta-2-trimethylsilylethyl glycoside of lacto-N-fucoheptaose based on a highly stereo- and regioselective glycosylation between a Lewis(x) trisaccharidic donor and a tetraol tetrasaccharidic acceptor. The title compound was characterized by high-resolution NMR spectroscopy.

Quantitative measurement of longitudinal and transverse cross-relaxation rates: an application to the analysis of the internal dynamics of ranalexin in water and trifluoroethanol.

We describe a quantitative processing method which gives access to the longitudinal and transverse cross-relaxation rates from off-resonance ROESY intensities. This method takes advantage of the dependence of the off-resonance ROESY experiments at any mixing time and any spin-lock angle θ on two relaxation matrices, the longitudinal and the transverse ones. This allows one to take into account multistep magnetization transfers even if the measurements are performed only at one or two mixing times. The ratio of the longitudinal to transverse cross-relaxation rates can then be used as a local indicator of the internal dynamics, without assuming a structure or a model of motion. After validation of this processing method by numerical simulations, it is applied to the analysis of the dynamics of the peptide ranalexin dissolved in pure water and in water/TFE.

NMR study of a Lewis(X) pentasaccharide derivative: solution structure and interaction with cations.

The structure and conformation of the synthetic pentasaccharide Gal(beta 1-4){Fuc(alpha 1-3)}GlcNAc(beta 1-3)Gal(beta 1-4)Glc-beta OMe of the Lewis(X) family has been determined by NMR spectroscopy in dimethyl sulfoxide and methanol. In these solvents, the binding constants with calcium have been evaluated as 9.5 and 29.6 M-1, respectively. Study of the interaction sites has been achieved through the use of paramagnetic divalent cations and distance triangulation methods. Two regions have been found, the first one in the vicinity of the fucose unit, the second one closer to the lactose part.

A method for determining B1 field inhomogeneity. Are the biases assumed in heteronuclear relaxation experiments usually underestimated?

We describe a method allowing the determination of the effective B1 field amplitude distribution in a high-resolution NMR spectrometer. This method which can be adapted to almost any sequence, essentially consists of a mutation followed by a purging B0 gradient pulse. Experimental results obtained with this approach are described in homonuclear and heteronuclear cases. The experimental distributions are used to estimate the biases induced by B1 inhomogeneity, as well as the loss of RF power on heteronuclear transverse self-relaxation rate determination. In this type of measurement, the experimental biases induced on the intensities can be as large as 5% for long mixing times.

Direct determination of the heteronuclear T1/T2 ratio by off-resonance steady-state magnetization measurement: Investigation of the possible application to fast exchange characterization of 15N-labeled proteins.

The (15)N steady-state magnetization in the presence of off-resonance rf irradiation is an analytical function of the T(1)/T(2) ratio and of the angle between the (15)N effective field axis and the static magnetic field direction. This relation holds whatever the relaxation mechanisms due to motions on the nanosecond time scale, and the size of the spin system. If motions on the micro- to millisecond time scale are present (fast exchange), the same observable depends also on their spectral density at the frequency of the effective field. The cross-peak intensity in each 2D (15)N-(1)H correlation map is directly related to the dynamic parameters, so that the characterization of fast exchange phenomena by this method is in principle less time-consuming than the separate measurement of self-relaxation rates. The theory of this approach is described. Its practical validity is experimentally evaluated on a (15)N-labeled 61 amino acid neurotoxin. It turns out that existing equipments lead to non-negligible biases. Their consequences for the accuracy attainable, at present, by this method are investigated in detail.

1H NMR spectroscopy study of the dynamic properties of glycogen in solution by steady-state magnetisation measurement with off-resonance irradiation.

The dynamics of size-selected fractions of glycogen in solution have been investigated by proton NMR spectroscopy, using a recently described relaxation study method which relies on strong offresonance irradiation. The dependence of the steady-state magnetisation on angle and intensity of the effective radio-frequency field was measured and compared to theoretical curves derived from different models of motion. Absence or presence of contributions to relaxation from molecular motions on the microsecond time scale can be tested with this method, without having to resort to models. We found that glycogen dipolar relaxation did not result from isotropic Brownian rotation, and despite some contribution from slow motion (> 1 microsecond) to relaxation in glycogen alpha-particles extracted from rat liver, bulk movement of the molecules did not appear to participate in averaging the dipolar term to zero. Whereas hepatic glycogen rat beta-particles and commercial oyster glycogen displayed very similar relaxation properties, alpha-particles showed significantly different behaviour. However, all results were compatible with a diversity of movements within the molecule, ranging from freely rotating pyranoside rings through collective chain motion and possibly to bulk movement of the beta sub-units within the alpha-particle.

Local mobility of 15N labeled biomolecules characterized through cross-correlation rates: Applications to paramagnetic proteins.

The mobility of (15)N labeled proteins can be characterized by measuring the cross-correlation rates delta(N,NI) that govern the conversion of Zeeman order N(z) of an amide (15)N nucleus into longitudinal two-spin order 2N(z)I(z) involving the amide (15)N and (1)H nuclei. This represents an alternative to the measurement of (15)N self-relaxation rates 1/T(1) and 1/T(2) or 1/T(1rho). The rate of interconversion between N(z) and 2N(z)I(z) is due to cross-correlation between fluctuations of different interactions and is not affected by a variety of relaxation mechanisms that contribute to the self-relaxation rates 1/T(1), 1/T(2) and 1/T(1rho). Spin diffusion among protons, which affects the measurements, can be quenched by various means that are evaluated by experiments and simulations. By applying an off-resonance radio-frequency (RF) field in the vicinity of the nitrogen resonance, the spectral density function J(omega) can be determined at the frequency origin and at the nitrogen Larmor frequency. The methods are applied to the paramagnetic High-Potential Iron-Sulfur Protein iso I (HiPIP I) from E. halophila in its reduced state.

Off-resonance rf fields in heteronuclear NMR: Application to the study of slow motions.

The advantages of using off-resonance rf fields in heteronuclear self-relaxation experiments are explored on a fully (15)N-enriched protein. It is firstly shown that in the absence of slow motions the longitudinal and transverse (15)N self-relaxation rate values derived with this method are in agreement with the ones measured by the classical inversion-recovery and Carr-Purcell-Meiboom-Gill (CPMG) sequences, respectively. Secondly, by comparing the (15)N transverse self-relaxation rates obtained by the proposed off-resonance sequence and by the CPMG sequence, 11 residues out of the 61 of toxin α are shown to exhibit a chemical exchange phenomenon in water on a time scale ranging from 1 µs to 100 ms. By varying the effective field amplitude, chemical exchange processes involving these residues are measured and the corresponding correlation times are evaluated without having assumed any motion model. Similar, though less precise, results are given by the analysis of the (15)N off-resonance self-relaxation rates on the basis of the Lipari-Szabo model to describe the fast internal dynamics of toxin α.

Solution structure of a Lewis(x) analogue by off-resonance 1H NMR spectroscopy without use of an internal distance reference.

A recent 1H NMR method has been applied to the determination of the solution structure and internal dynamics of a synthetic mixed C/O trisaccharide related to sialyl Lewis(x). Varying the rf field offset in ROESY-type experiments enabled the measurement of longitudinal and transverse dipolar cross-relaxation rates with high accuracy. Assuming that for each proton pair the motion could be represented by a single exponential autocorrelation function, it was possible to derive geometrical parameters (r) and dynamic parameters (tau(cp)). With this assumption, 224 cross-relaxation rates have been transformed into 30 interproton distance constraints and 30 dipolar correlation times. The distance constraints have been used in a simulated-annealing procedure. This trisaccharide exhibits a structure close to the O-glycosidic analogue, but its flexibility seems highly reduced. On the basis of the determined structure and dynamics, it is shown that no conformational exchange occurs, the molecule existing in the form of a unique family in aqueous solution. In order to assess the quality of the resulting structures and to validate this new experimental procedure of distance extraction, we finally compare these solution structures to the ones obtained using three different sets of distances deduced from three choices of internal reference. It appears that this procedure allows the determination of the most precise and accurate solution.

A self-included cyclomaltoheptaose derivative studied by NMR spectroscopy and molecular modelling.

The 3D structure of 6-deoxy-6-L-tyrosinylamidocyclomaltoheptaose, a self-complexing beta-cyclodextrin derivative, was determined by NMR and molecular modelling. The aminoacyl side-chain is included in the cavity and induces chemical-shift variations in the CD proton signals, allowing their complete assignment. Dipolar interactions between protons of the tyrosine ring and internal protons of the cyclodextrin were used to obtain distance constraints. Then 42 structures were calculated from 32 distance constraints--21 shorter than 4 A involve the host-guest interactions--using a simulated annealing procedure. Starting from one of the resulting structures, a 250-ps molecular dynamics simulation was carried out in a waterbox without constraint. The simulation data are in agreement with NMR data such as nOe and ring-current effects. The cyclodextrin part takes an elliptical shape, which tightly fits the aromatic moiety. As a consequence, the respective motion of the host and the guest moieties have the same amplitude and time scale: the self-inclusion complex shows only little flexibility.