High-resolution Nuclear Magnetic Resonance Techniques Research Articles

Characterization of Posttranslationally Modified PHF-1 Tau Peptides Using Gaussian Accelerated Molecular Dynamics Simulation.

The microtubule-associated protein, Tau, is an intrinsically disordered protein that plays a crucial role in neurodegenerative diseases like Alzheimer's disease. The posttranslational modifications across the Tau protein domains are involved in regulating Tau protein's function and disease onset. Of the various posttranslational modifications at Ser, Thr, and Tyr sites, O-GlcNAcylation and phosphorylation are the most critical ones, playing a vital role in Tau aggregation and tauopathies. To understand the function, it is essential to characterize the structural changes associated withTaumodification. Previous experimental studies have focused on high-resolution nuclear magnetic resonance techniques to structurally characterize the effect of phosphorylation, O-GlcNAcylation, and combination of both PTMs on Tau conformation in small peptides centered on the PHF-1 epitope from amino acid 392 to 411. The structural characterization using atomistic molecular dynamics simulation of such disordered peptides requires long simulation time, proper sampling method, and utilization of appropriate force fields for accurate determination of conformational ensembles, resembling the experimental data. This chapter details the protocol for the structural characterization of modified Tau peptides using the CHARMM36m force field and enhanced sampling methods like Gaussian accelerated molecular dynamics (GaMD) simulation. We have focused on a detailed explanation of the GaMD method and analyses of molecular dynamics trajectories to explain the relationship between two modifications, phospho- and glyco-, at C-terminus of Tau protein and its stable conformation over the longer simulation timeframes. The analyses involve energetics reweighting, clustering of simulation trajectories, and characterization of secondary structure using circular dichroism data from the simulation. The reader can utilize this protocol to investigate the structures of complex proteins, especially the disordered ones.

The potentiality of NMR-based metabolomics in food science and food authentication assessment.

In the last years, there was an increasing interest on nuclear magnetic resonance (NMR) spectroscopy, whose applications experienced an exponential growth in several research fields, particularly in food science. NMR was initially developed as the elective technique for structure elucidation of single molecules and nowadays is playing a dominant role in complex mixtures investigations. In the era of the "omics" techniques, NMR was rapidly enrolled as one of the most powerful methods to approach metabolomics studies. Its use in analytical routines, characterized by rapid and reproducible measurements, would provide the identification of a wide range of chemical compounds simultaneously, disclosing sophisticated frauds or addressing the geographical origin, as well as revealing potential markers for other authentication purposes. The great economic value of high-quality or guaranteed foods demands highly detailed characterization to protect both consumers and producers from frauds. The present scenario suggests metabolomics as the privileged approach of modern analytical studies for the next decades. The large potentiality of high-resolution NMR techniques is here presented through specific applications and using different approaches focused on the authentication process of some foods, like tomato paste, saffron, honey, roasted coffee, and balsamic and traditional balsamic vinegar of Modena, with a particular focus on geographical origin characterization, ageing determination, and fraud detection.

Sugar-Functional Vinyl Addition Poly(norbornene)-Photopatternable Poly(norbornenyl gluconamide) Compositions Developed with Water.

The norbornenyl gluconamide (NBGA) monomer can be polymerized by a number of palladium catalysts to give water-soluble, vinyl addition poly(NBGA). Depending on the catalyst used, the reaction conditions, and the chain-transfer additives employed, high-molecular-weight polymers can be obtained. These polymers can be thermally cross-linked at ca. 190 °C or at ca. 150 °C when the difunctional glutaraldehyde is added. A photopatternable composition is formed by the addition of a water-soluble diazide when the poly(NBGA) molecular weight is sufficiently high. After image-wise exposure, negative-tone patterns are revealed by water development. A detailed analysis of NBGA monomer structures by high-resolution nuclear magnetic resonance techniques including pure shift is reported showing that two diastereomers of each endo- and exo-isomer are formed from the reaction of norbornene methylamine and δ-gluconolactone.

Orientational order in liquid crystals by combiningH2andC13nuclear magnetic resonance spectroscopy and density functional theory calculations

Structural and orientational order properties of the liquid crystal 4,4'-bis-heptyl-azoxybenzene (HAB) have been obtained in its nematic and smectic- A phases by simultaneously analyzing several observables extracted from 2H and 13C nuclear magnetic resonance (NMR) spectra, i.e., 2H quadrupolar, 2H-1H and 13C-2H dipolar couplings, as well as 13C chemical shift anisotropy. 13C experiments required the application of high-resolution solid-state NMR techniques like 1H high-power decoupling and cross polarization, as well as the independent determination of chemical shift tensors by means of density functional theory (DFT) calculations, here performed taking into account the effect of the anisotropic medium by the polarizable continuum model method. The approach, consisting in the simultaneous analysis of all the 2H and 13C experimental data to derive orientational order parameters, and in the use of geometrical parameters determined by DFT methods, allows more detailed and reliable results to be obtained with respect to the traditional approach based on the sole analysis of 2H experiments.

NMR Study of Slow Motions of HDA Hydrocarbons Chains Inside Lamellar Structures

Measurements of 1H and 13C Nuclear Magnetic Resonance (NMR) for the nanocomposite materials formed by the intercalation of hexadecylamine (HDA) in metal oxides (TiO2, V2O5 and MoO3), are reported. The 1H NMR spin-lattice relaxation in the rotating frame was described by using the spectral density due to Davidson and Cole, which incorporates a distribution of correlation times characterized by a width parameter ϵ. The fitting of the data was obtained for ϵ = 0.74, indicating that the correlation times are distributed over a narrow range in this system. High-resolution 13C NMR techniques were used to resolve the NMR lines of middle-chain methylene groups in the spectra and variable contact time cross-polarization {1H-}13C experiments were employed to analyze the reorientation dynamics of the CH3 and CH2 groups in the HDA chains.

NMR Metabolomics of Planktonic and Biofilm Modes of Growth in Pseudomonas aeruginosa

Bacteria often reside in communities where the cells have secreted sticky, polymeric compounds that allow them to attach to surfaces. This sessile lifestyle, referred to as a biofilm, affords the cells within these communities a tolerance of antibiotics and antimicrobial treatments. Biofilms of the bacterium Pseudomonas aeruginosa have been implicated in cystic fibrosis and are capable of colonizing medical implant devices, such as heart valves and catheters, where treatment of the infection often requires the removal of the infected device. This mode of growth is in stark contrast to planktonic, free floating cells, which are more easily eradicated with antibiotics. The mechanisms contributing to a biofilm's tenacity and a planktonic cell's susceptibility are just beginning to be explored. In this study, we have used a metabolomic approach employing nuclear magnetic resonance (NMR) techniques to study the metabolic distinctions between these two modes of growth in P. aeruginosa. One-dimensional 1H NMR spectra of fresh growth medium were compared with spent medium supernatants from batch and chemostat planktonic and biofilms generated in continual flow system culture. In addition, 1H high-resolution magic angle spinning NMR techniques were employed to collect 1H NMR spectra of the corresponding cells. Principal component analysis and spectral comparisons revealed that the overall metabolism of planktonic and biofilm modes of growth appeared similar for the spent media, while the planktonic and biofilm cells displayed marked differences. To determine the robustness of this technique, we prepared cell samples under slightly different preparation methods. Both techniques showed similar results. These feasibility studies show that there exist chemical differences between planktonic and biofilm cells; however, in order to identify these metabolomic differences, more extensive studies would have to be performed, including 1H-1H total correlated spectroscopy.

Sexual conjugation in yeast: A paradigm to study G-protein-coupled receptor domain structure.

The yeast Saccharomyces cerevisiae undergoes cell fusion during sexual conjugation to form diploid cells. The haploids participating in this process signal each other through secreted peptide-mating factors (alpha-factor and a-factor) that are recognized by G-protein-coupled receptors. The receptor (Ste2p) recognizing the tridecapeptide alpha-factor is used as a model system in our laboratory to understand various aspects of peptide-receptor interactions and receptor structure. Using chemical procedures we have synthesized peptides corresponding to the seven transmembrane domains of Ste2p and studied their structures in membrane mimetic environments. Extension of these studies requires preparation of longer fragments of Ste2p. This article discusses strategies used in our laboratory to prepare peptides containing multiple domains of Ste2p. Data are presented on the use of chemical synthesis, biosynthesis, and native chemical ligation. Using biosynthetic approaches fusion proteins have been expressed that contain single receptor domains, two transmembrane domains connected by the contiguous loop, and the tail connected to the seventh transmembrane domain. Tens of milligrams of fusion protein were obtained per liter, and multimilligram quantities of the isotopically labeled target peptides were isolated using such biosynthetic approaches. Initial circular dichroism results on a chemically synthesized 64-residue peptide containing a portion of the cytosolic tail and the complete seventh transmembrane domain showed that the tail portion and the hydrophobic core of this peptide maintained individual conformational preferences. Moreover, this peptide could be studied at near millimolar concentrations in the presence of micelles and did not aggregate under these conditions. Thus, these constructs can be investigated using high-resolution nuclear magnetic resonance techniques, and the cytosolic tail of Ste2p can be used as a hydrophilic template to improve solubility of transmembrane peptides for structural analysis.

Dynamic structure of pharaonis phoborhodopsin (sensory rhodopsin II) and complex with a cognate truncated transducer as revealed by site-directed 13C solid-state NMR

We have recorded 13C nuclear magnetic resonance (NMR) spectra of [3- 13C]Ala, [1- 13C]Val-labeled pharaonis phoborhodopsin ( ppR or sensory rhodopsin II) incorporated into egg PC (phosphatidylcholine) bilayer, by means of site-directed high-resolution solid-state NMR techniques. Seven 13C NMR signals from transmembrane α-helices were resolved for [3- 13C]Ala- ppR at almost the same positions as those of bacteriorhodopsin (bR), except for the suppressed peaks in the loop regions in spite of the presence of at least three Ala residues. In contrast, 13C NMR signals from the loops were visible from [1- 13C]Val- ppR but their peak positions of the transmembrane α-helices are not always the same between ppR and bR. The motional frequency of the loop regions in ppR was estimated as 10 5 Hz in view of the suppressed peaks from [3- 13C]Ala- ppR due to interference with proton decoupling frequency. We found that conformation and dynamics of ppR were appreciably altered by complex formation with a cognate truncated transducer pHtr II (1–159). In particular, the C-terminal α-helix protruding from the membrane surface is involved in the complex formation and subsequent fluctuation frequency is reduced by one order of magnitude.

Liquid- and solid-state high-resolution NMR methods for the investigation of aging processes of silicone breast implants

To investigate aging processes of silicone gel breast implants, which may include migration of free unreacted material from the gel and rubber to local (e.g. connective tissue capsule) or distant sites in the body, chemical alteration of the polymer and infiltration of body compounds, various approaches of multinuclear nuclear magnetic resonance (NMR) experiments ( 29Si, 13C, 1H) were evaluated. While 29Si, 13C, and 1H solid-state magic angle spinning (MAS) NMR techniques performed on virgin and explanted envelopes of silicone prostheses provided only limited information, high-resolution liquid-state NMR techniques of CDCl 3 extracts were highly sensitive analytical tools for the detection of aging related changes in the materials. Using 2D 1H, 1H correlation spectroscopy (COSY) and 29Si, 1H heteronuclear multiple bond coherence (HMBC) experiments with gradient selection, it was possible to detect lipids (mainly phospholipids) as well as silicone oligomer species in explanted envelopes and gels. Silicone oligomers were also found in connective tissue capsules, indicating that cyclic polysiloxanes can migrate from intact implants to adjacent and distant sites. Furthermore, lipids can permeate the implant and modify its chemical composition.

New NMR developments for structural investigation of proton-bearing materials at different length scales

Abstract Solid state nuclear magnetic resonance (NMR) spectroscopy is a powerful tool to characterize the coordination sphere of the selectively observed nucleus in crystalline or amorphous materials. Recent developments of high-resolution NMR techniques for proton in the solid state open the way to obtaining a more accurate description of the structure of proton-bearing materials at different length scales. This can be achieved through the combined use of Lee–Goldburg homonuclear proton decoupling and proton spin-diffusion. In that scope we describe a new 3D experiment that allows direct probing of proton spin-diffusion process between resolved proton spectra obtained under homonuclear decoupling. We anticipate that this class of experiments will soon be used to study poorly crystalline hybrid materials as well as finely divided or porous inorganic materials.

Conformational preferences of the kappa-selective opioid agonist U50488. A combined molecular mechanics and nuclear magnetic resonance study.

The conformational preferences of the kappa-selective opioid agonist U50488 have been studied using MM2-87 calculations and nuclear magnetic resonance (NMR) spectroscopy. The calculations were performed for the protonated form with a dielectric constant of 80 and the unprotonated form with dielectric constants of 1.5 and 80. A systematic search found 72 stable conformers with certain consistent conformational preferences for some of the important dihedral angles. The preferred conformers proved to be compact structures stabilized by intramolecular attractive van der Waals interactions, though at least some of these appear to be electrostatically unfavorable. The conformation of U50488 was also examined in aqueous solution using one-dimensional (1D) and two-dimensional (2D) high-resolution 1H NMR techniques such as the interpretation of 1H-1H vicinal coupling constants, 1D and 2D nuclear Overhauser effect (NOE) experiments, and 2D correlated spectroscopy (COSY) experiments. Five crystallographic conformations were examined as well. There was generally good agreement between all three methods of conformational analysis. There appeared to be a reasonable geometrical agreement between the relatively rigid kappa-agonist (-)-ketazocine and a gauche conformer of U50488. The proposed pharmacophore is also consistent with other kappa-selective analogs of U50488 including one in which the peptide bond is incorporated into a lactam ring. The low affinity of U50488 for mu-receptors was attributed to its cyclohexane ring which occupies space not present in the nonselective (-)-ketazocine.

Capsule structure of Proteus mirabilis (ATCC 49565).

Proteus mirabilis 2573 (ATCC 49565) produces an acidic capsular polysaccharide which was shown from glycose analysis, carboxyl reduction, methylation, periodate oxidation, and the application of one dimensional and two-dimensional high-resolution nuclear magnetic resonance techniques to be a high-molecular-weight polymer of branched trisaccharide units composed of 2-acetamido-2-deoxy-D-glucose (N-acetyl-D-glucosamine), 2-acetamido-2,6-dideoxy-L-galactose (N-acetyl-L-fucosamine), and D-glucuronic acid, having the structure: [formula: see text] P. mirabilis 2573 also produces an O:6 serotype lipopolysaccharide in which the O-chain component has the same structure as the homologous capsular polysaccharide. This is the first report of a defined capsular polysaccharide in this bacterial genus.

29Si nuclear magnetic resonance of amorphous hydrogenated silicon and amorphous microcrystalline mixed-phase hydrogenated silicon.

$^{29}\mathrm{Si}$ nuclear magnetic resonance (NMR) spectra have been measured for amorphous hydrogenated silicon (a-Si:H) and amorphous microcrystalline mixed-phase hydrogenated silicon (\ensuremath{\mu}c-Si:H) using high-resolution solid-state NMR techniques; magic-angle sample spinning, dipolar decoupling, and cross polarization (CP). $^{1}\mathrm{H}$ NMR spectra have been also measured. The peak position of the $^{29}\mathrm{Si}$ signal observed with the CP technique correlates well with the line shape of $^{1}\mathrm{H}$ NMR spectra, which has been ascribed to a chemical shift due to bonding configurations not only in the first coordination sphere but also in the second- and further-nearest coordination spheres. Origins of the $^{29}\mathrm{Si}$ linewidth have also been discussed, the magnitude of each contribution being estimated. A narrow $^{29}\mathrm{Si}$ signal can be obtained without the CP technique only in \ensuremath{\mu}c-Si:H, which demonstrates that there are regions where the silicon network is mobile. Based on the NMR results, microscopic models of the structures of a-Si:H and \ensuremath{\mu}c-Si:H are presented.

Magnetic Moment of the Triton in Units of the Magnetic Moment of the Proton

High-resolution nuclear magnetic resonance techniques were used to perform a precise measurement of the ratio of the Larmor frequencies of tritons and protons in a sample of 20% tritiated water. The measured ratio OMEGA /sub T/ OMEGA /sub H/ is 1.066 639 75(2). From this frequency ratio and in estimate of the magnetic shielding correction, a value of in units of the proton moment. From this value and the hfs measurements of Kusch, and of Prodell and Kusch, one obtains a hfs anomaly of (-5.62 plus or minus 0.13)10/sup -6/ for tritium. This observed anomaly is compared with the theoretical predictions of Adains. The longitudinal relaxation time T/sub 1/ of the protons and tritons in the sample was measured and found to be 1.02 plus or minus 0.10 sec. and 0.83 plus or minus 0.10 sec., respectively. (auth)