skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


  • NSF Public Access
  • Search Results
  • Labeling of methyl groups: a streamlined protocol and guidance for the selection of 2H precursors based on molecular weight
Title: Labeling of methyl groups: a streamlined protocol and guidance for the selection of 2H precursors based on molecular weight
Abstract A streamlined one-day protocol is described to produce isotopically methyl-labeled protein with high levels of deuterium for NMR studies. Using this protocol, the D2O and2H-glucose content of the media and protonation level of ILV labeling precursors (ketobutyrate and ketovalerate) were varied. The relaxation rate of the multiple-quantum (MQ) state that is present during the HMQC-TROSY pulse sequence was measured for different labeling schemes and this rate was used to predict upper limits of molecular weights for various labeling schemes. The use of deuterated solvents (D2O) or deuterated glucose is not required to obtain1H–13C correlated NMR spectra of a 50 kDa homodimeric protein that are suitable for assignment by mutagenesis. High quality spectra of 100–150 kDa proteins, suitable for most applications, can be obtained without the use of deuterated glucose. The proton on the β-position of ketovalerate appears to undergo partial exchange with deuterium under the growth conditions used in this study.  more » « less
Award ID(s):
2112872
PAR ID:
10570079
Author(s) / Creator(s):
; ;
Publisher / Repository:
Springer
Date Published:
Journal Name:
Journal of Biomolecular NMR
Volume:
78
Issue:
3
ISSN:
0925-2738
Page Range / eLocation ID:
149 to 159
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; (, Frontiers in Plant Science)
    The commonly used analytical tools for metabolomics cannot directly probe metabolic activities or distinguish metabolite differences between cells and suborgans in multicellular organisms. These issues can be addressed byin-vivoisotope labeling and mass spectrometry imaging (MSI), respectively, but the combination of the two, a newly emerging technology we call MSIi, has been rarely applied to plant systems. In this study, we explored MSIiofArabidopsis thalianawith D2O labeling to study and visualize D-labeling in three classes of lipids: arabidopsides, chloroplast lipids, and epicuticular wax. Similar to other stress responses, D2O-induced stress increased arabidopsides in an hour, but it was relatively minor for matured plants and reverted to the normal level in a few hours. The D-labeling isotopologue patterns of arabidopsides matched with those of galactolipid precursors, supporting the currently accepted biosynthesis mechanism. Matrix-assisted laser desorption/ionization (MALDI)-MSI was used to visualize the spatiotemporal distribution of deuterated chloroplast lipids, pheophytina, MGDGs, and DGDGs, after growing day-after-sowing (DAS) 28 plants in D2O condition for 3–12 days. There was a gradual change of deuteration amount along the leaf tissues and with a longer labeling time, which was attributed to slow respiration leading to low D2O concentration in the tissues. Finally, deuterium incorporation in epicuticular wax was visualized on the surfaces of the stem and flower. The conversion efficiency of newly synthesized C30 aldehyde to C29 ketone was very low in the lower stem but very high at the top of the stem near the flower or on the flower carpel. This study successfully demonstrated that MSIican unveil spatiotemporal metabolic activities in various tissues ofA. thaliana. 
    more » « less
  2. ; ; ; ; ; ; ; (, Advanced Science)
    Abstract Deuterium (D) labeling is of great value in organic synthesis, pharmaceutical industry, and materials science. However, the state‐of‐the‐art deuteration methods generally require noble metal catalysts, expensive deuterium sources, or harsh reaction conditions. Herein, noble metal‐free and ultrathin ZnIn2S4(ZIS) is reported as an effective photocatalyst for visible light‐driven reductive deuteration of carbonyls to produce deuterated alcohols using heavy water (D2O) as the sole deuterium source. Defective two‐dimensional ZIS nanosheets (D‐ZIS) are prepared in a surfactant assisted bottom‐up route exhibited much enhanced performance than the pristine ZIS counterpart. A systematic study is carried out to elucidate the contributing factors and it is found that the in situ surfactant modification enabled D‐ZIS to expose more defect sites for charge carrier separation and active D‐species generation, as well as high specific surface area, all of which are beneficial for the desirable deuteration reaction. This work highlights the great potential in developing low‐cost semiconductor‐based photocatalysts for organic deuteration in D2O, circumventing expensive deuterium reagents and harsh conditions. 
    more » « less
  3. ; ; ; ; (, The Astrophysical Journal)
    Abstract Direct D-H exchange in radicals is investigated in a quasi-uniform flow employing chirped-pulse millimeter-wave spectroscopy. Inspired by the H-atom catalyzed isomerization of C3H2reported in our previous study, D-atom reactions with the propargyl (C3H3) radical and its photoproducts were investigated. We observed very efficient D-atom enrichment in the photoproducts through an analogous process of D addition/H elimination to C3H2isomers occurring at 40 K or below. Cyclic C3HD is the only deuterated isomer observed, consistent with the expected addition/elimination yielding the lowest energy product. The other expected addition/elimination product, deuterated propargyl, is not directly detected, although its presence is inferred by the observations in the latter part of the flow. There, in the high-density region of the flow, we observed both isotopomers of singly deuterated propyne attributed to stabilization of the H+C3H2D or D+C3H3adducts. The implications of these observations for the deuterium fractionation of hydrocarbon radicals in astrochemical environments is discussed with the support of a monodeuterated chemical kinetic model. 
    more » « less
  4. ; ; ; ; (, Angewandte Chemie International Edition)
    Abstract Intermediates relevant to cobalt‐catalyzed alkene hydroformylation have been isolated and evaluated in fundamental organometallic transformations relevant to aldehyde formation. The 18‐electron (R,R)‐(iPrDuPhos)Co(CO)2H has been structurally characterized, and it promotes exclusive hydrogenation of styrene in the presence of 50 bar of H2/CO gas (1:1) at 100 °C. Deuterium‐labeling studies established reversible 2,1‐insertion of styrene into the Co−D bond of (R,R)‐(iPrDuPhos)Co(CO)2D. Whereas rapid β‐hydrogen elimination from cobalt alkyls occurred under an N2atmosphere, alkylation of (R,R)‐(iPrDuPhos)Co(CO)2Cl in the presence of CO enabled the interception of (R,R)‐(iPrDuPhos)Co(CO)2C(O)CH2CH2Ph, which upon hydrogenolysis under 4 atm H2produced the corresponding aldehyde and cobalt hydride, demonstrating the feasibility of elementary steps in hydroformylation. Both the hydride and chloride derivatives, (X=H−, Cl−), underwent exchange with free13CO. Under reduced pressure, (R,R)‐(iPrDuPhos)Co(CO)2Cl underwent CO dissociation to form (R,R)‐(iPrDuPhos)Co(CO)Cl. 
    more » « less
  5. ; ; ; ; ; ; (, Israel Journal of Chemistry)
    Abstract The T1of a hyperpolarized site in solution is a key parameter that determines the time‐window in which its NMR signals are observable. For13C sites adjacent to protons,1H‐decoupling has been shown to increase the hyperpolarized signal resolution and SNR. Additionally, polarization transfer to protons has shown utility in increasing the sensitivity of detection. However,1H‐decoupling could lead to a change in the decay rate of a hyperpolarized13C site. Here we tested this possible effect in a case where the protons are directly bound to an sp3hyperpolarized13C site (using [1,2‐13C2]choline) and1H‐decoupling was applied continuously throughout the hyperpolarized decay measurement. We found that1H‐decoupling did not lead to any significant changes in the13C polarization decay time but did result in the expected collapse of J‐coupling and produced sharper signals. This result suggested that1H‐decoupling did not affect the decay rate of hyperpolarized sp3 13C sites. The deuterium‐substitution approach (using [1,1,2,2‐D4,1‐13C]choline) showed a dramatic prolongation of T1. Upper bounds on the T1of all investigated sites were calculated. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email