Monday, August 22, 2011

Meet the Bridge12 Team

The Bridge12 team is looking forward to attending the following meetings this fall:

You can meet our team to discuss our lates innovations in gyrotron technology for DNP-enhanced NMR spectroscopy (DNP-NMR).

Operation of a Continuously Frequency-Tunable Second-Harmonic CW 330-GHz Gyrotron for Dynamic Nuclear Polarization

Torrezan, A.C., et al., Operation of a Continuously Frequency-Tunable Second-Harmonic CW 330-GHz Gyrotron for Dynamic Nuclear Polarization. IEEE Transactions on Electron Devices, 2011. 58(8): p. 2777-83.

The design and the operation of a frequency-tunable continuous-wave (CW) 330-GHz gyrotron oscillator operating at the second harmonic of the electron cyclotron frequency are reported. The gyrotron has generated 18 W of power from a 10.1 kV 190 mA electron beam working in a TE-4,3 cylindrical mode, corresponding to an efficiency of 0.9 %. The measured start oscillation current over a range of magnetic field values is in good agreement with theoretical start currents obtained from linear theory for successive high-order axial modes TE-4,3,q, where q = 1-6. Moreover, the observed frequency range in the start current measurement is in reasonable agreement with the frequency range obtained from numerical simulations. The minimum start current was measured to be 33 mA. A continuous tuning range of 1.2 GHz was experimentally observed via a combination of magnetic, voltage, and thermal tuning. The gyrotron output power and frequency stabilities were assessed to be 0.4% and 3 ppm, respectively, during a 110-h uninterrupted CW run. Evaluation of the gyrotron output microwave beam pattern using a pyroelectric camera indicated a Gaussian-like mode content of 92% with an ellipticity of 28%. The gyrotron will be used for 500-MHz nuclear magnetic resonance experiments with sensitivity enhanced by dynamic nuclear polarization.

Tuesday, August 16, 2011

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

Bayro, M. J., Debelouchina, G. T., Eddy, M. T., Birkett, N. R., MacPhee, C. E., Rosay, M., Maas, W. E., Dobson, C. M., and Griffin, R. G. (2011) Intermolecular Structure Determination of Amyloid Fibrils with Magic-Angle Spinning and Dynamic Nuclear Polarization NMR, J. Am. Chem. Soc.

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 13C-13C correlation spectra and the third based on the identification of intermolecular interactions in 13C-15N spectra. We show, in studies of fibrils formed by the 86-residue SH3 domain of PI3 kinase (PI3-SH3 or PI3K-SH3), that efficient 13C-13C 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 13C-13C experiments are corroborated by 15N-13C correlation spectra obtained from a mixed [15N,12C]/[14N,13C] 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 15N-13C 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 13C-13C and 15N-13C constraints that establish that the PI3-SH3 protein strands are aligned in a parallel, in-register arrangement within the amyloid fibril.

Para-hydrogen induced polarization of amino acids, peptides and deuterium-hydrogen gas

This article is about para-hydrogen induced polarization, a technique related to microwave/terahertz driven polarization enhancement.

Signal Amplification by Reversible-Exchange (SABRE) is a method of hyperpolarizing substrates by polarization transfer from para-hydrogen without hydrogenation. Here, we demonstrate that this method can be applied to hyperpolarize small amounts of all proteinogenic amino acids and some chosen peptides down to the nanomole regime and can be detected in a single scan in low-magnetic fields down to 0.25 mT (10 kHz proton frequency). An outstanding feature is that depending on the chemical state of the used catalyst and the investigated amino acid or peptide, hyperpolarized hydrogen-deuterium gas is formed, which was detected with 1H and 2H NMR spectroscopy at low magnetic fields of B0 = 3.9 mT (166 kHz proton frequency) and 3.2 mT (20 kHz deuterium frequency).

Hyperpolarized [1-13C]-Ascorbic and Dehydroascorbic Acid: Vitamin C as a Probe for Imaging Redox Status in Vivo

Bohndiek, S. E., Kettunen, M. I., Hu, D.-e., Kennedy, B. W. C., Boren, J., Gallagher, F. A., and Brindle, K. M. (2011) Hyperpolarized [1-13C]-Ascorbic and Dehydroascorbic Acid: Vitamin C as a Probe for Imaging Redox Status in Vivo, J. Am. Chem. Soc. 133, 11795-11801.

Dynamic nuclear polarization (DNP) of 13C-labeled metabolic substrates in vitro and their subsequent intravenous administration allow both the location of the hyperpolarized substrate and the dynamics of its subsequent conversion into other metabolic products to be detected in vivo. We report here the hyperpolarization of [1-13C]-ascorbic acid (AA) and [1-13C]-dehydroascorbic acid (DHA), the reduced and oxidized forms of vitamin C, respectively, and evaluate their performance as probes of tumor redox state. Solution-state polarization of 10.5 ± 1.3% was achieved for both forms at pH 3.2, whereas at pH 7.0, [1-13C]-AA retained polarization of 5.1 ± 0.6% and [1-13C]-DHA retained 8.2 ± 1.1%. The spin‚Äìlattice relaxation times (T1's) for these labeled nuclei are long at 9.4 T: 15.9 ± 0.7 s for AA and 20.5 ± 0.9 s for DHA. Extracellular oxidation of [1-13C]-AA and intracellular reduction of [1-13C]-DHA were observed in suspensions of murine lymphoma cells. The spontaneous reaction of DHA with the cellular antioxidant glutathione was monitored in vitro and was approximately 100-fold lower than the rate observed in cell suspensions, indicating enzymatic involvement in the intracellular reduction. [1-13C]-DHA reduction was also detected in lymphoma tumors in vivo. In contrast, no detectable oxidation of [1-13C]-AA was measured in the same tumors, consistent with the notion that tumors maintain a reduced microenvironment. This study demonstrates that hyperpolarized 13C-labeled vitamin C could be used as a noninvasive biomarker of redox status in vivo, which has the potential to translate to the clinic.

Beyond the Silica Surface by Direct Silicon-29 Dynamic Nuclear Polarization

Lafon, O., Rosay, M., Aussenac, F., Lu, X., Trébosc, J., Cristini, O., Kinowski, C., Touati, N., Vezin, H., and Amoureux, J.-P. (2011) Beyond the Silica Surface by Direct Silicon-29 Dynamic Nuclear Polarization, Angew. Chem. Int. Ed., n/a-n/a.

Buried truth: High-field magic angle spinning dynamic nuclear polarization (MAS DNP) enhances the sensitivity of solid-state NMR spectroscopy, but only for protonated surfaces. Direct 29Si DNP using the biradical TOTAPOL circumvents this limitation by producing a 30-fold enhancement of subsurface 29Si NMR signals in mesoporous silica, a material with applications in photonics, nanotechnology and catalysis.