• Title/Summary/Keyword: /sup n/C-NMR

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Triple isotope-[13C, 15N, 2H] labeling and NMR measurements of the inactive, reduced monomer form of Escherichia coli Hsp33

  • Lee, Yoo-Sup;Ko, Hyun-Suk;Ryu, Kyoung-Seok;Jeon, Young-Ho;Won, Hyung-Sik
    • Journal of the Korean Magnetic Resonance Society
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    • v.14 no.2
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    • pp.117-126
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    • 2010
  • Hsp33 is a molecular chaperone achieving a holdase activity upon response to a dual stress by heat and oxidation. Despite several crystal structures available, the activation process is not clearly understood, because the structure inactive Hsp33 as its reduced, zinc-bound, monomeric form has not been solved yet. Thus, we initiated structural investigation of the reduced Hsp33 monomer by NMR. In this study, to overcome the high molecular weight (33 kDa), the protein was triply isotope-[$^{13}C$, $^{15}N$, $^2H$]-labeled and its inactive, monomeric state was ensured. 2D-[$^1H$, $^{15}N$]-TROSY and a series of triple resonance spectra could be successfully obtained on a high-field (900 MHz) NMR machine with a cryoprobe. However, under all of the different conditions tested, the number of resonances observed was significantly less than that expected from the amino acid sequence. Thus, a possible contribution of dynamic conformational exchange leading to a line broadening is suggested that might be important for activation process of Hsp33.

Paraelectric-Ferroelectric Phase Transition of (NH4)2SO4 Single Crystals by 14N NMR

  • Lim, Ae Ran
    • Journal of the Korean Magnetic Resonance Society
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    • v.21 no.2
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    • pp.63-66
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    • 2017
  • The $^{14}N$ NMR spectra for $(NH_4)_2SO_4$ crystals were obtained near the phase transition temperature $T_C=223K$, and were found to precisely reflect the symmetry change in the crystal at this first-order phase transition. Changes in the resonance frequencies near $T_C$ were attributed to the structural phase transition. In the ferroelectric and paraelectric phases, two inequivalent NH4 groups were distinguished in the $^{14}N$ NMR spectra. The two types, $NH_4$(1) and $NH_4$(2), have slightly different local environments. Consequently, we conclude that the phase transition is caused by the change in the environment of the $^{14}N$ nuclei in the $NH_4$ groups, rather than by the $SO_4$ groups.

Secondary Structure, 1H, 13C and 15N Resonance Assignments and Molecular Interactions of the Dishevelled DIX Domain

  • Capelluto, Daniel G.S.;Overduin, Michael
    • BMB Reports
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    • v.38 no.2
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    • pp.243-247
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    • 2005
  • Dishevelled (Dvl) is a positive regulator of the canonical Wnt signaling pathway, which regulates the levels of $\beta$-catenin. The $\beta$-catenin oncoprotein depends upon the association of Dvl and Axin proteins through their DIX domains, and its accumulation directs the expression of specific developmental-related genes at the nucleus. Here, the $^1H$, $^{13}C$, and $^{15}N$ resonances of the human Dishevelled 2 DIX domain are assigned using heteronuclear nuclear magnetic resonance (NMR) spectroscopy. In addition, helical and extended elements are identified based on the NMR data. The results establish a structural context for characterizing the actin and phospholipid interactions and binding sites of this novel domain, and provide insights into its role in protein localization to stress fibers and cytoplasmic vesicles during Wnt signaling.

Backbone 1H, 15N, and 13C Resonance Assignment of HP1242 from Helicobacter pylori

  • Kang, Su-Jin;Park, Sung-Jean;Jung, Seo-Jeong;Lee, Bong-Jin
    • BMB Reports
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    • v.38 no.5
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    • pp.591-594
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    • 2005
  • One of the small proteins from Helicobacter pylori, HP1242, was investigated by the solution nuclear magnetic resonance (NMR) spectroscopy. HP1242 is known as a 76-residue conserved hypothetical protein and its function cannot be identified based on sequence homology. Here, the results of the backbone $^1H$, $^{15}N$, and $^{13}C$ resonance assignments of the HP1242 are reported using double- and triple-resonance techniques. About 95% of all of the $^1HN$, $^{15}N$, $^{13}CO$, $^{13}C{\alpha}$, and $^{13}C{\beta}$ resonances that cover 75 non- Proline residues of the 76 residues are clarified through sequential- and specific- assignments. In addition, three helical regions were clearly identified on the basis of the resonance assignments.

Chemical Analysis and Isolation of Antibacterial Compound from Ulmus Species (II) : Isolation and Chemical Structure of Antibacterial Compound (느릅나무 근피의 화학조성분 및 항균성 물질(II) : 항균성 물질의 단리 및 화학구조)

  • Kim, Chang-Soon;Lee, Jung-Myoung;Choi, Chang-Ok;Park, Soung-Bae;Eom, Tae-Jin
    • Journal of the Korean Wood Science and Technology
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    • v.31 no.1
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    • pp.16-21
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    • 2003
  • The methanol extractives from root bark of Ulmus davidiana var japonica nakai were fractionated with n-hexane, ethyl ether, ethyl acetate and waster, the water soluble fraction was also fractionated with silicagel column chromatograhy. The chemical structure of purifided compounds were identified with UV, IR, 1H-NMR and 13C-NMR spectra and the antibacterial activities also were investigated. Two different antibacterial compounds (compound A and B) were fractionated with silicagel chromatography and TLC. Compounds B was identified as a catechin rahmnoside. The both of compounds had antibacterial activity on Staphylococcus aureus and Salmonella typhimurium.

Backbone 1H, 15N, and 13C Resonance Assignments and Secondary-Structure of the Conserved Hypothetical Protein HP0892 of Helicobacter pylori

  • Han, Kyung-Doo;Park, Sung-Jean;Jang, Sun-Bok;Lee, Bong-Jin
    • Molecules and Cells
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    • v.25 no.1
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    • pp.138-141
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    • 2008
  • HP0892 (SwissProt/TrEMBL ID O25552) is a 90-residue conserved hypothetical protein from Helicobacter pylori strain 26695, with a calculated pI of 9.38 and a molecular mass of 10.41 kDa. It belongs to the Plasmid stabilization system protein family (PF05016) in the Pfam database. Proteins with sequence similarity to HP0892 exist in Vibrio choierae, Enterococcus faecalis, Campylobacter jejuni, Streptococcus pneumoniae, Haemophilus influenzae, Escherichia coli O157. Here we report the sequence-specific backbone resonance assignments of HP0892 using multidimentional heteronuclear NMR spectroscopy. About 97.0% (422/435) of the HN, N, CO, $C{\beta}$, $C{\alpha}$ resonances of 90 residues of HP0892 were assigned. On the basis of the resonance assignments, three helical regions and four strand regions were identified using the CSI program. This study is a prerequisite for calculating the solution structure of HP0892, and will be useful for studying its interaction with other molecules.

Cost-effective isotope labeling technique developed for 15N/13C-labeled proteins

  • Kim, Hee-Youn;Hong, Eun-Mi;Lee, Weon-Tae
    • Journal of the Korean Magnetic Resonance Society
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    • v.15 no.2
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    • pp.115-127
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    • 2011
  • A newly developed cost-effective approach to prepare $^{15}N/^{13}C$-labeled protein for NMR studies is presented. This method has been successfully applied to isotopically labeling of PTK6 SH2 domain and MTH 1880 protein. The production method generates cell density using a growing media containing $^{15}NH_4Cl$, $^{12}C_6$-D-glucose. Following a doubling time period for unlabeled metabolite exhaustion and then addition $^{13}C_6$-D-glucose into a M9 growing media, the cells are induced. Our results demonstrate that in order to get full incorporation of $^{13}C$, the isotopes are not totally required during the initial growth phase before induction. The addition of small amounts of $^{13}C_6$-D-glucose to the induction phase is sufficient to obtain more than 95% incorporation of isotopes into the protein. Our optimized protocol is two-thirds less costly than the classical method using $^{13}C$ isotope during the entire growth phase.

Sensitivity Enhancement of Methyl-TROSY by Longitudinal 1H Relaxation Optimization

  • Lee, Dong-Han;Vijayan, Vinesh;Montaville, Pierre;Becker, Stefan;Griesinger, Christian
    • Journal of the Korean Magnetic Resonance Society
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    • v.13 no.1
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    • pp.15-26
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    • 2009
  • The NMR detection of methyl groups is of keen interest because they provide the long-range distance information required to establish global folds of high molecular weight proteins. Using longitudinal $^1H$ relaxation optimization, we achieve a gain in sensitivity of approximately 1.6-fold in the methyl-TROSY and its NOESY experiments for the 38 kDa protein mitogen activated protein kinase p38 in its fully protonated and $^{13}C$ and $^{15}N$ labeled state.

Use of Correlation of 1H and 13C Chemical Shifts of N-Arylsuccinanilic Acids,N-Arylsuccinimides, N-Arylmaleanilic Acids, and N-Arylmaleimides with the Hammett Substituent Constants for the Studies of Electronic Effects

  • Lee, Hye-Sun;Yu, Ji-Sook;Lee, Chang-Kiu
    • Bulletin of the Korean Chemical Society
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    • v.30 no.10
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    • pp.2351-2354
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    • 2009
  • A series of N-arylsuccinanilic acids, N-arylsuccinimides, N-arylmaleanilic acids, and N-arylmaleimides was prepared and their NMR spectra were examined by correlating the $^1H\;and\;^{13}C$ chemical shift values with the corresponding Hammett $\sigma$ values. The carbonyl carbons of the amides show a normal correlation with $\sigma$ but those of the imides show an inverse correlation.