Simulation of oriented NMR spectra: Combining molecular dynamics and chemical shift tensor calculations.

Solid state NMR is widely used to study the orientation and other structural features of proteins and peptides in lipid bilayers. Using data obtained by PISEMA (Polarization Inversion Spin Exchange at Magic Angle) experiments, periodic spectral patterns arise from well-aligned α-helical molecules. Significant problems in the interpretation of PISEMA spectra may arise for systems that do not form perfectly defined secondary structures, like α-helices, or the signal pattern is disturbed by molecular motion. Here, we present a new method that combines molecular dynamics simulation with tensorial orientational constraints (MDOC) and chemical shift tensor calculations for the simulation and interpretation of PISEMA-like spectra. The calculations include the spectra arising from non α-helical molecules and molecules with non-uniform intrinsic mobility. In a first step, dipolar or quadrupolar interaction tensors drive molecular rotations and reorientations to obtain the proper mean values as observed in corresponding NMR experiments. In a second step, the coordinate snapshots of the MDOC simulations are geometry optimized with the isotropic 15 N chemical shifts as constraints using Bond Polarization Theory (BPT) to provide reliable 15 N CS tensor data. The averaged dipolar 1 H-15 N couplings and the δzz tensor components can then be combined to simulate PISEMA patterns. We apply this method to the ß-helical peptide gramicidin A (gA) and demonstrate that this method enables the assignment of most PISEMA resonances. In addition, MDOC simulations provide local order parameters for the calculated sites. These local order parameters reveal large differences in backbone mobility between L- and D-amino acids of gA.

Development of in situ high resolution NMR: Proof-of-principle for a new (spinning) cylindrical mini-pellet approach applied to a Lithium ion battery.

Solid-state nuclear magnetic resonance (ssNMR) spectroscopy is a powerful technique for characterizing the local structure and dynamics of battery and other materials. It has been widely used to investigate bulk electrode compounds, electrolytes, and interfaces. Beside common ex situ investigations, in situ and operando techniques have gained considerable importance for understanding the reaction mechanisms and cell degradation of electrochemical cells. Herein, we present the recent development of in situ magic angle spinning (MAS) NMR methodologies to study batteries with high spectral resolution, setting into context possible advances on this topic. A mini cylindrical cell type insert for 4 mm MAS rotors is introduced here, being demonstrated on a Li/VO2F electrochemical system, allowing the acquisition of high-resolution 7Li MAS NMR spectra, spinning the electrochemical cell up to 15 kHz.

Structures Controlled by Entropy: The Flexibility of Strychnine as Example.

To study the flexibility of strychnine, we performed molecular dynamics simulations with orientational tensorial constraints (MDOC). Tensorial constraints are derived from nuclear magnetic resonance (NMR) interaction tensors, for instance, from residual dipolar couplings (RDCs). Used as orientational constraints, they rotate the whole molecule and molecular parts with low rotational barriers. Since the NMR parameters are measured at ambient temperatures, orientational constraints generate conformers that populate the whole landscape of Gibbs free energy. In MDOC, structures are populated that are not only controlled by energy but by the entropy term TΔS of the Gibbs free energy. In the case of strychnine, it is shown that ring conformers are populated, which has not been discussed in former investigations. These conformer populations are not only in accordance with RDCs but fulfill nuclear Overhauser effect (NOE)-derived distance constraints and 3JHH couplings as well.

Screening of Nutraceuticals and Plant Extracts for Inhibition of Amyloid-ß Fibrillation.

Fluorescence spectroscopy for in vitro amyloid-ß (Aß) fibrillation diagnosis and spectral fluorescence signature for the identification of bioactive compounds were applied to study traditional Ayurvedic nutraceuticals - Brahmi, Ashwagandha, Shanka pushpi, and Gotu kola - as well as their plant extracts for possible treatment of Alzheimer's disease. All samples manifest as inhibitors on three different variants of the Aß peptide: methionine Aß1-40, Aß1-40, and Aß1-42. The main compounds within the nutraceuticals were identified. Since related medicals are known to have reduced negative post- and side-effects and even may introduce further positive health impacts by preventing pathogen plaque formation and reducing free Aß to a natural level, such treatment approaches could be of further interest.

Investigation of backbone dynamics and local geometry of bio-molecules using calculated NMR chemical shifts and anisotropies.

Prerequisite for chemical shift (CS) and CS tensor calculations are highly refined structures defining the molecular surroundings of the nuclei under study. Here, we present geometry optimizations with 13C and 15N CS constraints for large bio-molecules like peptides and proteins. The method discussed here provides both, refined structures and chemical shift tensors. Furthermore, since the experimental resonances of aligned systems are related to CS tensors, they strongly depend on the orientation and motion of molecules, their fragments, functional groups and moieties. For efficient CS calculations we apply a semi-empirical approach-the bond polarization theory (BPT). The BPT relies on linear bond polarization parameters and we present a new set of parameters based on ab initio second-order Møller-Plesset perturbation theory calculations. The new parametrization extends the applicability of the BPT approach to a wide range of organic molecules and bio-polymers. Here, the method has been applied to the protein ubiquitin and the membrane-active peptide gramicidin A (dimer) in oriented bilayers. The calculated 13C and 15N CS values of best-refined structures published until now gave a large scatter with respect to the experiment. It will be shown that BPT CS optimizations can reduce these errors to values near the experimental uncertainty. In combination with molecular dynamics with orientational constraints it is possible to study motional dynamics and BPT calculations can provide residual chemical shift anisotropies.

Synthesis of Fast Fluoride-Ion-Conductive Fluorite-Type Ba1- xSb xF2+ x (0.1 ≤ x ≤ 0.4): A Potential Solid Electrolyte for Fluoride-Ion Batteries.

Toward the development of high-performance solid electrolytes for fluoride-ion batteries, fluorite-type nanostructured solid solutions of Ba1- xSb xF2+ x ( x ≤ 0.4) were synthesized by high-energy ball-milling method. Substitution of divalent Ba2+ by trivalent Sb3+ leads to an increase in interstitial fluoride-ion concentration, which enhances the ionic conductivity of the Ba1- xSb xF2+ x (0.1 ≤ x ≤ 0.4) system. Total ionic conductivities of 4.4 × 10-4 and 3.9 × 10-4 S cm-1 were obtained for Ba0.7Sb0.3F2.3 and Ba0.6Sb0.4F2.4 compositions at 160 °C, respectively. In comparison to isostructural Ba0.3La0.7F2.3, the ionic conductivity of Ba0.7Sb0.3F2.3 is significantly higher, which is attributed to the presence of an electron lone pair on Sb3+. Introduction of such lone pairs seems to increase fluoride-ion mobility in solid solutions. In addition, Ba0.7Sb0.3F2.3 was tested as a cathode material against Ce and Zn anode using La0.9Ba0.1F2.9 as the electrolyte. Ba0.3Sb0.7F2.3/La0.9Ba0.1F2.9/Ce cell showed high discharge and charge capacities of 301 and 170 mA h g-1, respectively, in the first cycle at 150 °C.

1H line width dependence on MAS speed in solid state NMR - Comparison of experiment and simulation.

Recent developments in magic angle spinning (MAS) technology permit spinning frequencies of ≥100 kHz. We examine the effect of such fast MAS rates upon nuclear magnetic resonance proton line widths in the multi-spin system of ß-Asp-Ala crystal. We perform powder pattern simulations employing Fokker-Plank approach with periodic boundary conditions and 1H-chemical shift tensors calculated using the bond polarization theory. The theoretical predictions mirror well the experimental results. Both approaches demonstrate that homogeneous broadening has a linear-quadratic dependency on the inverse of the MAS spinning frequency and that, at the faster end of the spinning frequencies, the residual spectral line broadening becomes dominated by chemical shift distributions and susceptibility effects even for crystalline systems.

Medical Plants and Nutraceuticals for Amyloid-ß Fibrillation Inhibition.

Plaque formation due to amyloid-ß oligomerization and fibrillation is a key issue for its deposition in the brains of dementia and Alzheimer's disease patients. Related drugs preventing this peptide fibril accumulation bear the potential of considerable medical and social value. In this study, we performed in vitro fibrillation inhibition tests with eight different medical plant extracts and nutraceuticals using fluorescence spectroscopy. Successful inhibition of the following plant extracts and nutraceuticals were obtained: Withania somnifera, Centella asiatica, Bacopa monnieri, and Convolvulus pluricaulis, providing new drug candidates for the prevention and treatment of Alzheimer's disease.

Molecular dynamics simulations on PGLa using NMR orientational constraints.

NMR data obtained by solid state NMR from anisotropic samples are used as orientational constraints in molecular dynamics simulations for determining the structure and dynamics of the PGLa peptide within a membrane environment. For the simulation the recently developed molecular dynamics with orientational constraints technique (MDOC) is used. This method introduces orientation dependent pseudo-forces into the COSMOS-NMR force field. Acting during a molecular dynamics simulation these forces drive molecular rotations, re-orientations and folding in such a way that the motional time-averages of the tensorial NMR properties are consistent with the experimentally measured NMR parameters. This MDOC strategy does not depend on the initial choice of atomic coordinates, and is in principle suitable for any flexible and mobile kind of molecule; and it is of course possible to account for flexible parts of peptides or their side-chains. MDOC has been applied to the antimicrobial peptide PGLa and a related dimer model. With these simulations it was possible to reproduce most NMR parameters within the experimental error bounds. The alignment, conformation and order parameters of the membrane-bound molecule and its dimer were directly derived with MDOC from the NMR data. Furthermore, this new approach yielded for the first time the distribution of segmental orientations with respect to the membrane and the order parameter tensors of the dimer systems. It was demonstrated the deuterium splittings measured at the peptide to lipid ratio of 1/50 are consistent with a membrane spanning orientation of the peptide.

Li(+) intercalation in isostructural Li2VO3 and Li2VO2F with O(2-) and mixed O(2-)/F(-) anions.

Mixed-anion materials for Li-ion batteries have been attracting attention in view of their tunable electrochemical properties. Herein, we compare two isostructural (Fm3̅m) model intercalation materials Li2VO3 and Li2VO2F with O(2-) and mixed O(2-)/F(-) anions, respectively. Synchrotron X-ray diffraction and pair distribution function data confirm large structural similarity over long-range and at the atomic scale for these materials. However, they show distinct electrochemical properties and kinetic behaviour arising from the different anion environments and the consequent difference in cationic electrostatic repulsion. In comparison with Li2VO3 with an active V(4+/5+) redox reaction, the material Li2VO2F with oxofluoro anions and the partial activity of V(3+/5+) redox reaction favor higher theoretical capacity (460 mA h g(-1)vs. 230 mA h g(-1)), higher voltage (2.5 V vs. 2.2 V), lower polarization (0.1 V vs. 0.3 V) and faster Li(+) chemical diffusion (∼10(-9) cm(2) s(-1)vs. ∼10(-11) cm(2) s(-1)). This work not only provides insights into the understanding of anion chemistry, but also suggests the rational design of new mixed-anion battery materials.

Solid electrolytes for fluoride ion batteries: ionic conductivity in polycrystalline tysonite-type fluorides.

Batteries based on a fluoride shuttle (fluoride ion battery, FIB) can theoretically provide high energy densities and can thus be considered as an interesting alternative to Li-ion batteries. Large improvements are still needed regarding their actual performance, in particular for the ionic conductivity of the solid electrolyte. At the current state of the art, two types of fluoride families can be considered for electrolyte applications: alkaline-earth fluorides having a fluorite-type structure and rare-earth fluorides having a tysonite-type structure. As regard to the latter, high ionic conductivities have been reported for doped LaF3 single crystals. However, polycrystalline materials would be easier to implement in a FIB due to practical reasons in the cell manufacturing. Hence, we have analyzed in detail the ionic conductivity of La(1-y)Ba(y)F(3-y) (0 ≤ y ≤ 0.15) solid solutions prepared by ball milling. The combination of DC and AC conductivity analyses provides a better understanding of the conduction mechanism in tysonite-type fluorides with a blocking effect of the grain boundaries. Heat treatment of the electrolyte material was performed and leads to an improvement of the ionic conductivity. This confirms the detrimental effect of grain boundaries and opens new route for the development of solid electrolytes for FIB with high ionic conductivities.

Multilayered core-shell structure of polyol-stabilized calcium fluoride nanoparticles characterized by NMR.

Diethyleneglycol (DEG)-stabilized calcium fluoride (CaF(2)) nanoparticles with a mean diameter of about 20 nm consist of an inorganic crystalline core, covered by a roughly monomolecular layer of organic material according to standard physico-chemical analysis. Multinuclear NMR experiments under magic angle spinning were used to identify the constituent (13)C, (1)H and (19)F atoms. All resonances were assigned (DEG, EtOH, H(2)O, H(3)O(+), OH(-), F(-), CaF(2)), and different populations could be discriminated in terms of their mobilities and correlations with neighboring atoms. We observed several different layers: mobile EtOH on the particle surface, an "interphase" containing immobile DEG molecules and fluoride ions, plus the single-crystalline CaF(2) core. It was thus possible for the first time to characterize by NMR all layered components of such core-shell particles, and the thickness of the newly discovered interphase could be estimated from the NMR intensities.

Calculation of fluorine chemical shift tensors for the interpretation of oriented (19)F-NMR spectra of gramicidin A in membranes.

A semi-empirical method for the prediction of chemical shifts, based on bond polarization theory, has recently been introduced for (13)C. Here, we extended this approach to calculate the (19)F chemical shift tensors of fluorine bound to aromatic rings and in aliphatic CF(3) groups. For the necessary parametrization, ab initio chemical shift calculations were performed at the MP2 level for a set of fluorinated molecules including tryptophan. The bond polarization parameters obtained were used to calculate the (19)F chemical shift tensors for several crystalline molecules, and to reference the calculated values on a chemical shift scale relative to CFCl(3). As a first biophysical application, we examined the distribution of conformations of a (19)F-labeled tryptophan side chain in the membrane-bound ion channel peptide, gramicidin A. The fluorine chemical shift tensors were calculated from snapshots of a molecular dynamics simulation employing the (19)F-parametrized bond polarization theory. In this MD simulation, published (2)H quadrupolar and (15)N-(1)H dipolar couplings of the indole ring were used as orientational constraints to determine the conformational distribution of the 5F-Trp(13) side chain. These conformations were then used to interpret the spectra of (19)F-labeled gramicidin A in fluid and gel phase lipid bilayers.

Spectral assignments and anisotropy data of cellulose I(alpha): 13C-NMR chemical shift data of cellulose I(alpha) determined by INADEQUATE and RAI techniques applied to uniformly 13C-labeled bacterial celluloses of different Gluconacetobacter xylinus strains.

Solid-state (13)C-NMR spectroscopy was used to characterize native cellulose pellicles from two strains of Gluconacetobacter xylinus (ATCC 53582, ATCC 23769), which had been statically cultivated in Hestrin-Schramm (HS) medium containing fully (13)C-labeled beta-D-glucose-U-(13)C(6) as the sole source of carbon. For both samples, the (13)C-NMR chemical shifts were completely assigned for each (13)C-labeled site of cellulose I(alpha) with the aid of 2D refocused INADEQUATE NMR. To determine the principal chemical shift tensor components, a pulse sequence based on the recoupling of anisotropy information (RAI) was applied at 10 kHz MAS. The detailed (13)C tensors of cellulose I(alpha) from different bacterial celluloses are thus available now for the first time, and these results have been compared with previously published data of nonenriched material and with theoretical predictions.

Solid-state 19F NMR spectroscopy reveals that Trp41 participates in the gating mechanism of the M2 proton channel of influenza A virus.

The integral membrane protein M2 of influenza A virus assembles as a tetrameric bundle to form a proton-conducting channel that is activated by low pH. The side chain of His37 in the transmembrane alpha-helix is known to play an important role in the pH activation of the proton channel. It has also been suggested that Trp41, which is located in an adjacent turn of the helix, forms part of the gating mechanism. Here, a synthetic 25-residue peptide containing the M2 transmembrane domain was labeled with 6F-Trp41 and studied in lipid membranes by solid-state 19F NMR. We monitored the pH-dependent differences in the 19F dipolar couplings and motionally narrowed chemical shift anisotropies of this 6F-Trp41 residue, and we discuss the pH activation mechanism of the H+ channel. At pH 8.0, the structural parameters implicate an inactivated state, while at pH 5.3 the tryptophan conformation represents the activated state. With the aid of COSMOS force field simulations, we have obtained new side-chain torsion angles for Trp41 in the inactivated state (chi1 = -100 degrees +/- 10 degrees , chi2 = +110 degrees +/- 10 degrees ), and we predict a most probable activated state with chi1 = -50 degrees +/- 10 degrees and chi2 = +115 degrees +/- 10 degrees . We have also validated the torsion angles of His37 in the inactivated state as chi1 = -175 degrees +/- 10 degrees and chi2 = -170 degrees +/- 10 degrees .

Solid state 19F NMR parameters of fluorine-labeled amino acids. Part I: aromatic substituents.

Structural parameters of peptides and proteins in biomembranes can be directly measured by solid state NMR of selectively labeled amino acids. The 19F nucleus is a promising label to overcome the low sensitivity of 2H, 13C or 15N, and to serve as a background-free reporter group in biological compounds. To make the advantages of solid state 19F NMR fully available for structural studies of polypeptides, we have systematically measured the chemical shift anisotropies and relaxation properties of the most relevant aromatic and aliphatic 19F-labeled amino acids. In this first part of two consecutive contributions, six different 19F-substituents on representative aromatic side chains were characterized as polycrystalline powders by static and MAS experiments. The data are also compared with results on the same amino acids incorporated in synthetic peptides. The spectra show a wide variety of lineshapes, from which the principal values of the CSA tensors were extracted. In addition, temperature-dependent T(1) and T(2) relaxation times were determined by 19F NMR in the solid state, and isotropic chemical shifts and scalar couplings were obtained in solution.

Low-E probe for (19)F-(1)H NMR of dilute biological solids.

Sample heating induced by radio frequency (RF) irradiation presents a significant challenge to solid state NMR experiments in proteins and other biological systems, causing the sample to dehydrate which may result in distorted spectra and a damaged sample. In this work we describe a large volume, low-E (19)F-(1)H solid state NMR probe, which we developed for the 2D (19)F CPMG studies of dilute membrane proteins in a static and electrically lossy environment at 600MHz field. In (19)FCPMG and related multi-pulse (19)F-(1)H experiments the sample is heated by the conservative electric fields E produced in the sample coil at both (19)F and (1)H frequencies. Instead of using a traditional sample solenoid, our low-E (19)F-(1)H probe utilizes two orthogonal loop-gap resonators in order to minimize the conservative electric fields responsible for sample heating. Absence of the wavelength effects in loop-gap resonators results in homogeneous RF fields and enables the study of large sample volumes, an important feature for the dilute protein preparations. The orthogonal resonators also provide intrinsic isolation between the (19)F and (1)H channels, which is another major challenge for the (19)F-(1)H circuits where Larmor frequencies are only 6% apart. We detail steps to reduce (19)F background signals from the probe, which included careful choice of capacitor lubricants and manufacture of custom non-fluorinated coaxial cables. Application of the probe for two-dimensional (19)F CPMG spectroscopy in oriented lipid membranes is demonstrated with Flufenamic acid (FFA), a non-steroidal anti-inflammatory drug.

All-atom molecular dynamics simulations using orientational constraints from anisotropic NMR samples.

Orientational constraints obtained from solid state NMR experiments on anisotropic samples are used here in molecular dynamics (MD) simulations for determining the structure and dynamics of several different membrane-bound molecules. The new MD technique is based on the inclusion of orientation dependent pseudo-forces in the COSMOS-NMR force field. These forces drive molecular rotations and re-orientations in the simulation, such that the motional time-averages of the tensorial NMR properties approach the experimentally measured parameters. The orientational-constraint-driven MD simulations are universally applicable to all NMR interaction tensors, such as chemical shifts, dipolar couplings and quadrupolar interactions. The strategy does not depend on the initial choice of coordinates, and is in principle suitable for any flexible molecule. To test the method on three systems of increasing complexity, we used as constraints some deuterium quadrupolar couplings from the literature on pyrene, cholesterol and an antimicrobial peptide embedded in oriented lipid bilayers. The MD simulations were able to reproduce the NMR parameters within experimental error. The alignment of the three membrane-bound molecules and some aspects of their conformation were thus derived from the NMR data, in good agreement with previous analyses. Furthermore, the new approach yielded for the first time the distribution of segmental orientations with respect to the membrane and the order parameter tensors of all three systems.

NMR chemical shift powder pattern recoupling at high spinning speed and theoretical tensor evaluation applied to silk fibroin.

The NMR pulse sequence RAI (recoupling of anisotropy information) has been improved to obtain powder patterns at high MAS spinning speeds. The 2D iso-aniso experiment displays the static chemical shift spectra on the indirect dimension and the MAS spectra on the direct dimension; hence overlapping chemical shift tensor patterns can be well resolved. This efficient technique is applicable to compounds containing (13)C sp(3) (C(alpha), C(beta)) and sp(2) (C=O) sites with higher chemical shift (CS) anisotropy (CSA), and the reliability of the method was tested here on the (13)C chemical shift tensors of polycrystalline glycine, alanine, and serine. Subsequently, the same experiment was applied to the native silk protein fibroin from Bombyx mori, which consists mainly of these three amino acids. Molecular dynamics (MD) simulations of the silk II crystal structure of Takahashi et al. (Takahashi et al. Int. J. Biol. Macromol. 1999, 24, 127-138) were carried out to study the influence of motions on the chemical shift tensors. The (13)C chemical shift tensors were calculated using the bond polarization theory BPT on 200 structures created by an MD simulation. Very good agreement of the theoretical chemical shift anisotropy values with the experimental NMR results was obtained. The tensor orientations in the protein structure could thus be reliably derived.

Chemical shift driven geometry optimization.

A new method for refinement of 3D molecular structures by geometry optimization is presented. Prerequisites are a force field and a very fast procedure for the calculation of chemical shifts in every step of optimization. To the energy, provided by the force field (COSMOS force field), a pseudoenergy, depending on the difference between experimental and calculated chemical shifts, is added. In addition to the energy gradients, pseudoforces are computed. This requires the derivatives of the chemical shifts with respect to the coordinates. The pseudoforces are analytically derived from the integral expressions of the bond polarization theory. Single chemical shift values attributed to corresponding atoms are considered for structural correction. As a first example, this method is applied for proton position refinement of the D-mannitol X-ray structure. A crystal structure refinement with 13C chemical shift pseudoforces is carried out.