Dynamic Nuclear Polarization-Enhanced Solid-State NMR Spectroscopy of GNNQQNY Nanocrystals and Amyloid Fibrils

Dynamic nuclear polarization (DNP) utilizes the inherently larger polarization of electrons to enhance the sensitivity of conventional solid-state NMR experiments at low temperature. Recent advances in instrumentation development and sample preparation have transformed this field and have opened up new opportunities for its application to biological systems. Here, we present DNP-enhanced [superscript 13]C–[superscript 13]C and [superscript 15]N–[superscript 13]C correlation experiments on GNNQQNY nanocrystals and amyloid fibrils acquired at 9.4 T and 100 K and demonstrate that DNP can be used to obtain assignments and site-specific structural information very efficiently. We investigate the influence of temperature on the resolution, molecular conformation, structural integrity and dynamics in these two systems. In addition, we assess the low-temperature performance of two commonly used solid-state NMR experiments, proton-driven spin diffusion (PDSD) and transferred echo double resonance (TEDOR), and discuss their potential as tools for measurement of structurally relevant distances at low temperature in combination with DNP.National Institutes of Health (U.S.) (Grant EB002804)National Institutes of Health (U.S.) (Grant EB003151)National Institutes of Health (U.S.) (Grant EB002026

Intermolecular Structure Determination of Amyloid Fibrils with 2 Magic-Angle Spinning and Dynamic Nuclear Polarization NMR

We describe magic-angle spinning NMR experiments designed to elucidate the interstrand architecture of amyloid fibrils. Three methods are introduced for this purpose, two being based on the analysis of long-range [superscript 13]C–[superscript 13]C correlation spectra and the third based on the identification of intermolecular interactions in [superscript 13]C–[superscript 15]N spectra. We show, in studies of fibrils formed by the 86-residue SH3 domain of PI3 kinase (PI3-SH3 or PI3K-SH3), that efficient [superscript 13]C–[superscript 13]C correlation spectra display a resonance degeneracy that establishes a parallel, in-register alignment of the proteins in the amyloid fibrils. In addition, this degeneracy can be circumvented to yield direct intermolecular constraints. The [superscript 13]C–[superscript 13]C experiments are corroborated by [superscript 15]N–[superscript 13]C correlation spectra obtained from a mixed [[superscript 15]N,[superscript 12]C]/[[superscript 14]N,[superscript 13]C] sample which directly quantify interstrand distances. Furthermore, when the spectra are recorded with signal enhancement provided by dynamic nuclear polarization (DNP) at 100 K, we demonstrate a dramatic increase (from 23 to 52) in the number of intermolecular [superscript 15]N–[superscript 13]C constraints detectable in the spectra. The increase in the information content is due to the enhanced signal intensities and to the fact that dynamic processes, leading to spectral intensity losses, are quenched at low temperatures. Thus, acquisition of low temperature spectra addresses a problem that is frequently encountered in MAS spectra of proteins. In total, the experiments provide 111 intermolecular [superscript 13]C–[superscript 13]C and [superscript 15]N–[superscript 13]C constraints that establish that the PI3-SH3 protein strands are aligned in a parallel, in-register arrangement within the amyloid fibril.National Institutes of Health (U.S.) (Grant EB-003151)National Institutes of Health (U.S.) (Grant EB-002804)National Institutes of Health (U.S.) (Grant EB-002026

Solid-State Dynamic Nuclear Polarization at 263 GHz: Spectrometer Design and Experimental Results

Dynamic Nuclear Polarization (DNP) experiments transfer polarization from electron spins to nuclear spins with microwave irradiation of the electron spins for enhanced sensitivity in nuclear magnetic resonance (NMR) spectroscopy. Design and testing of a spectrometer for magic angle spinning (MAS) DNP experiments at 263 GHz microwave frequency, 400 MHz 1H frequency is described. Microwaves are generated by a novel continuous-wave gyrotron, transmitted to the NMR probe via a transmission line, and irradiated on a 3.2 mm rotor for MAS DNP experiments. DNP signal enhancements of up to 80 have been measured at 95 K on urea and proline in water–glycerol with the biradical polarizing agent TOTAPOL. We characterize the experimental parameters affecting the DNP efficiency: the magnetic field dependence, temperature dependence and polarization build-up times, microwave power dependence, sample heating effects, and spinning frequency dependence of the DNP signal enhancement. Stable system operation, including DNP performance, is also demonstrated over a 36 h period.National Institutes of Health (U.S.) (NIH grant EB-002804)National Institutes of Health (U.S.) (NIH grant EB-002026

Sensitivity-enhanced nuclear magnetic resonance of biological solids

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2001.Includes bibliographical references.Dynamic Nuclear Polarization (DNP) enhances the sensitivity of solid-state Nuclear Magnetic Resonance experiments via transfer of polarization from electrons to nuclei. The polarization transfer is driven by microwave irradiation near the electron Larmor frequency. Signal enhancements of up to approx. 185 at 5 Tesla have previously been reported using a nitroxide radical in water/glycerol and a high-power gyrotron as a microwave source. This thesis describes the continuation of these experiments with a focus towards developing methodology for structural studies of biological solids. DNP experiments are typically performed at low temperatures since the DNP efficiency increases with the electronic and nuclear spin-lattice relaxation times. Many biological systems, especially membrane proteins, have relatively short nuclear relaxation times even at 10 K. DNP experiments on fd bacteriophage and purple membrane show that significant sensitivity enhancements can still be obtained on systems with short relaxation times. DNP-enhanced spectra of the coat protein and DNA of fd bacteriophage demonstrate that proton spin diffusion evenly distributes the enhanced polarization throughout a large macromolecular assembly.(cont.) High-Field DNP can be combined with Magic Angle Spinning (MAS) for high-resolution spectroscopy. The gyrotron has been redesigned to enable MAS/DNP experiments at temperatures approaching liquid nitrogen. The DNP efficiency was investigated as a function of a number of different parameters and signal enhancements of up to approx. 20 at 90 K are reported. The initial two-dimensional chemical shift correlation spectra obtained with DNP at high field illustrate the stability of the gyrotron and low-temperature spinning apparatus.by Melanie Madeleine Rosay.Ph.D

Highly Efficient, Water-Soluble Polarizing Agents for Dynamic Nuclear Polarization at High Frequency

International audienceWell polarized: Two new polarizing agents PyPol and AMUPol soluble in glycerol/water mixtures are used for dynamic nuclear polarization (DNP) NMR spectroscopy. The enhancement factors (ε) are about 3.5 to 4 times larger than for the established agent TOTAPOL at 263 and 395 GHz. For AMUPol, the temperature dependence of ε allows DNP experiments to be performed at temperatures significantly higher than for typical high-field DNP NMR experiments

Sensitivity-enhanced solid-state NMR detection of expansin\u27s target in plant cell walls

Structure determination of protein binding to noncrystalline macromolecular assemblies such as plant cell walls (CWs) poses a significant structural biology challenge. CWs are loosened during growth by expansin proteins, which weaken the noncovalent network formed by cellulose, hemicellulose, and pectins, but the CW target of expansins has remained elusive because of the minute amount of the protein required for activity and the complex nature of the CW. Using solid-state NMR spectroscopy, combined with sensitivity-enhancing dynamic nuclear polarization (DNP) and differential isotopic labeling of expansin and polysaccharides, we have now determined the functional binding target of expansin in the Arabidopsis thaliana CW. By transferring the electron polarization of a biradical dopant to the nuclei, DNP allowed selective detection of 13C spin diffusion from trace concentrations of 13C, 15N-labeled expansin in the CW to nearby polysaccharides. From the spin diffusion data of wild-type and mutant expansins, we conclude that to loosen the CW, expansin binds highly specific cellulose domains enriched in xyloglucan, whereas more abundant binding to pectins is unrelated to activity. Molecular dynamics simulations indicate short 13C-13C distances of 4-6 A between a hydrophobic surface of the cellulose microfibril and an aromatic motif on the expansin surface, consistent with the observed NMR signals. DNP-enhanced 2D 13C correlation spectra further reveal that the expansin-bound cellulose has altered conformation and is enriched in xyloglucan, thus providing unique insight into the mechanism of CW loosening. DNP-enhanced NMR provides a powerful, generalizable approach for investigating protein binding to complex macromolecular targets

Effect of natriuretic peptides, a mammalian hormone, on whole bacterial Pseudomonas aeruginosa proteomic expression through nanoLC-ESI-MS/MS approach

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Brute-Force Hyperpolarization for NMR and MRI

Hyperpolarization (HP) of nuclear spins is critical for ultrasensitive nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI). We demonstrate an approach for >1500-fold enhancement of key small-molecule metabolites: 1-¹³C-pyruvic acid, 1-¹³C-sodium lactate, and 1-¹³C-acetic acid. The ¹³C solution NMR signal of pyruvic acid was enhanced 1600-fold at <i>B</i> = 1 T and 40 °C by pre-polarizing at 14 T and ∼2.3 K. This “brute-force” approach uses only field and temperature to generate HP. The noted 1 T observation field is appropriate for benchtop NMR and near the typical 1.5 T of MRI, whereas high-field observation scales enhancement as 1/<i>B</i>. Our brute-force process ejects the frozen, solid sample from the low-<i>T</i>, high-<i>B</i> polarizer, passing it through low field (<i>B</i> < 100 G) to facilitate “thermal mixing”. That equilibrates ¹H and ¹³C in hundreds of milliseconds, providing ¹³C HP from ¹H Boltzmann polarization attained at high <i>B</i>/<i>T</i>. The ejected sample arrives at a room-temperature, permanent magnet array, where rapid dissolution with 40 °C water yields HP solute. Transfer to a 1 T NMR system yields ¹³C signals with enhancements at 80% of ideal for noted polarizing conditions. High-resolution NMR of the same product at 9.4 T had consistent enhancement plus resolution of ¹³C shifts and <i>J</i>-couplings for pyruvic acid and its hydrate. Comparable HP was achieved with frozen aqueous lactate, plus notable enhancement of acetic acid, demonstrating broader applicability for small-molecule NMR and metabolic MRI. Brute-force avoids co-solvated free-radicals and microwaves that are essential to competing methods. Here, unadulterated samples obviate concerns about downstream purity and also exhibit slow solid-state spin relaxation, favorable for transporting HP samples