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Dynamic nuclear polarization for sensitivity enhancement in modern solid-state NMR
Jul 24, 2017 - 10:10 AM - by nmrlearner
nmrlearner's Avatar Dynamic nuclear polarization for sensitivity enhancement in modern solid-state NMR


Publication date: Available online 23 July 2017
Source:Progress in Nuclear Magnetic Resonance Spectroscopy

Author(s): Aany Sofia Lilly Thankamony, Johannes J. Wittmann, Monu Kaushik, Björn Corzilius

The field of dynamic nuclear polarization has undergone tremendous developments and diversification since its inception more than 6 decades ago. In this review we will provide an in-depth overview of the relevant topics involved in DNP-enhanced MAS NMR spectroscopy. This includes the theoretical description of DNP mechanisms as well as of the polarization transfer pathways that can lead to a uniform or selective spreading of polarization between nuclear spins. Furthermore, we will cover historical and state-of-the art aspects of dedicated instrumentation, polarizing agents, and optimization techniques for efficient MAS DNP. Finally, we present an extensive overview on applications in the fields of structural biology and materials science, that underlines that MAS DNP has moved far beyond the proof-of-concept stage and has become an important tool for research in these fields. Edited by Geoffrey Bodenhausen and Beat Meier
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[NMR paper] Recombinant expression of Ixolaris, a Kunitz-type inhibitor from the tick salivary gland, for NMR studies.
Jul 24, 2017 - 10:10 AM - by nmrlearner
nmrlearner's Avatar Recombinant expression of Ixolaris, a Kunitz-type inhibitor from the tick salivary gland, for NMR studies.

Related Articles Recombinant expression of Ixolaris, a Kunitz-type inhibitor from the tick salivary gland, for NMR studies.

Protein Expr Purif. 2017 Jul 19;:

Authors: De Paula VS, Silva FHS, Francischetti IMB, Monteiro RQ, Valente AP

Abstract
Ixolaris is an anticoagulant protein identified in the tick saliva of Ixodes scapularis. Ixolaris contains 2 Kunitz like domains and binds to Factor Xa or Factor X as a scaffold for inhibition of the Tissue Factor (TF)/Factor VIIa (FVIIa). In contrast to tissue factor pathway inhibitor (TFPI), however, Ixolaris does not bind to the active site cleft of FXa. Instead, complex formation is mediated by the FXa heparin-binding exosite. Due to its potent and long-lasting antithrombotic activity, Ixolaris is a promising agent for anticoagulant therapy. Although numerous functional studies of Ixolaris exist, three-dimensional structure of Ixolaris has not been obtained at atomic resolution. Using the pET32 vector, we successfully expressed a TRX-His6-Ixolaris fusion protein. By combining Ni-NTA chromatography, enterokinase protease cleavage, and reverse phase HPLC (RP-HPLC), we purified isotopically labeled Ixolaris for NMR studies. 1D (1)H and 2D (15)N-(1)H NMR analysis yielded high quality 2D (15)N-(1)H HSQC spectra revealing that the recombinant protein is folded. These studies represent the first steps in obtaining... [Read More]
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[NMR paper] Expanding the horizons for structural analysis of fully protonated protein assemblies by NMR spectroscopy at MAS frequencies above 100*kHz.
Jul 24, 2017 - 10:10 AM - by nmrlearner
nmrlearner's Avatar Expanding the horizons for structural analysis of fully protonated protein assemblies by NMR spectroscopy at MAS frequencies above 100*kHz.

Related Articles Expanding the horizons for structural analysis of fully protonated protein assemblies by NMR spectroscopy at MAS frequencies above 100*kHz.

Solid State Nucl Magn Reson. 2017 Jul 03;:

Authors: Struppe J, Quinn CM, Lu M, Wang M, Hou G, Lu X, Kraus J, Andreas LB, Stanek J, Lalli D, Lesage A, Pintacuda G, Maas W, Gronenborn AM, Polenova T

Abstract
The recent breakthroughs in NMR probe technologies resulted in the development of MAS NMR probes with rotation frequencies exceeding 100*kHz. Herein, we explore dramatic increases in sensitivity and resolution observed at MAS frequencies of 110-111*kHz in a novel 0.7*mm HCND probe that enable structural analysis of fully protonated biological systems. Proton- detected 2D and 3D correlation spectroscopy under such conditions requires only 0.1-0.5*mg of sample and a fraction of time compared to conventional (13)C-detected experiments. We discuss the performance of several proton- and heteronuclear- ((13)C-,(15)N-) based correlation experiments in terms of sensitivity and resolution, using a model microcrystalline fMLF tripeptide. We demonstrate the applications of ultrafast MAS to a large, fully protonated protein assembly of the 231-residue HIV-1 CA capsid protein. Resonance assignments of protons and heteronuclei, as well as (1)H-(15)N dipolar and (1)H(N) CSA tensors are... [Read More]
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[NMR paper] Reconstitution of Low-Density Lipoproteins with Fatty Acids for the Targeted Delivery of Drugs into Cancer Cells
Jul 24, 2017 - 10:10 AM - by nmrlearner
nmrlearner's Avatar Reconstitution of Low-Density Lipoproteins with Fatty Acids for the Targeted Delivery of Drugs into Cancer Cells


Low-density lipoproteins (LDLs) are a class of nanocarriers for the targeted delivery of therapeutics into aberrant cells that overexpress the LDL receptor. A facile procedure is used for reconstituting the hydrophobic core of LDLs with a binary fatty acid mixture. Facilitated by the tumor targeting capability of the apolipoprotein, the reconstituted, drug-loaded LDLs can effectively target cancer cells that overexpress the LDL receptor while showing minor adverse impact on normal fibroblasts. According to a hypothesized mechanism, the reconstituted LDLs can also enable metabolism-triggered drug release while preventing the payloads from lysosomal degradation. This study demonstrates that LDLs reconstructed with fatty acids hold great promise to serve as effective and versatile nanocarriers for targeted cancer therapy.Tumor-targeting nanocarriers: The hydrophobic core of native low-density lipoproteins (LDLs) can be reconstituted with naturally occurring fatty acids (see picture). This process facilitates metabolism-triggered drug release for targeted cancer therapy.

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Application Note: Efficient Uniform Labeling of Proteins Expressed in Baculovirus-Infected Insect Cells Using ... - SelectScience
Jul 23, 2017 - 11:38 AM - by nmrlearner
nmrlearner's Avatar Application Note: Efficient Uniform Labeling of Proteins Expressed in Baculovirus-Infected Insect Cells Using ... - SelectScience



Application Note: Efficient Uniform Labeling of Proteins Expressed in Baculovirus-Infected Insect Cells Using ...
SelectScience
Uniform isotope labeling is a key tool for NMR studies on recombinant proteins and their interaction with ligands of pharmaceutical interest. To be useful for most important NMR studies, a labeled protein has to be expressed with baculovirus-infected ...

and more »
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[NMR paper] NMR studies of large proteins.
Jul 22, 2017 - 9:28 PM - by nmrlearner
nmrlearner's Avatar NMR studies of large proteins.

Related Articles NMR studies of large proteins.

J Mol Biol. 2017 Jul 17;:

Authors: Jiang Y, Kalodimos CG

Abstract
Recent breakthroughs in isotope-labeling and pulse sequence techniques have enabled the Nuclear Magnetic Resonance (NMR) characterization of large protein systems with molecular weights of hundreds of kDa. NMR studies of a great variety of large proteins have provided unique insights into the binding, dynamic, and allosteric mechanisms. Here we present a brief summary of these developments by highlighting few cases that exemplify the uniqueness of NMR in providing atomic-resolution information into key dynamic processes and structures of protein complexes with high degree of flexibility.


PMID: 28728982 [PubMed - as supplied by publisher]



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Dynamic nuclear polarization-magnetic resonance imaging at low ESR irradiation frequency for ascorbyl free radicals
Jul 22, 2017 - 5:57 AM - by nmrlearner
nmrlearner's Avatar From The DNP-NMR Blog:

Dynamic nuclear polarization-magnetic resonance imaging at low ESR irradiation frequency for ascorbyl free radicals

p.p1 {margin: 0.0px 0.0px 0.0px 36.0px; text-indent: -36.0px; font: 12.0px Helvetica}
Ito, S. and F. Hyodo, Dynamic nuclear polarization-magnetic resonance imaging at low ESR irradiation frequency for ascorbyl free radicals. Scientific Reports, 2016. 6: p. 21407.


http://dx.doi.org/10.1038/srep21407


Highly water-soluble ubiquinone-0 (CoQ0) reacts with ascorbate monoanion (Asc) to mediate the production of ascorbyl free radicals (AFR). Using aqueous reaction mixture of CoQ0 and Asc, we obtained positively enhanced dynamic nuclear polarization (DNP)-magnetic resonance (MR) images of the AFR at low frequency (ranging from 515 to 530 MHz) of electron spin resonance (ESR) irradiation. The shape of the determined DNP spectrum was similar to ESR absorption spectra with doublet spectral peaks. The relative locational relationship of spectral peaks in the DNP spectra between the AFR (520 and 525 MHz), 14N-labeled carbamoyl-PROXYL (14N-CmP) (526.5 MHz), and Oxo63 (522 MHz) was different from that in the X-band ESR spectra, but were similar to that in the 300-MHz ESR spectra. The ratio of DNP enhancement to radical concentration for the AFR was higher than those for 14N-CmP, Oxo63, and flavin semiquinone radicals. The spectroscopic DNP properties observed for the AFR were essentially the same as those for AFR mediated by pyrroloquinoline quinone. Moreover, we made a success of in vivo DNP-MR imaging of the... [Read More]
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