Related ArticlesBiosynthetic 15N and 13C isotope labelling of glutathione in the mixed disulfide with Escherichia coli glutaredoxin documented by sequence-specific NMR assignments.
Eur J Biochem. 1993 Dec 1;218(2):327-34
Authors: Bushweller JH, Holmgren A, Wüthrich K
A biosynthetic procedure for obtaining 13C-15N doubly labelled glutathione from readily available precursor molecules is described. Isolation of the mutant Escherichia coli [C14S]glutaredoxin from E. coli cultures grown on 15N-13C doubly labelled media in the absence of reducing agents yields the mixed disulfide labelled in both the protein and the glutathione. 15N NMR assignments for glutathione obtained from two-dimensional [15N,1H]-correlation spectroscopy (COSY), and 13C NMR assignments for the entire mixed disulfide obtained from combined use of three-dimensional ct-HA[CAN]HN experiments and HCCH-total correlation spectroscopy ([HCCH]-TOCSY) demonstrated unequivocally that the glutathione is uniformly labelled with both 15N and 13C. This result also supports earlier suggestions that the intracellular glutaredoxin activity is sensitive to the glutathione redox status of the cell. Complete sets of 1H, 13C and 15N chemical shifts of both components in the mixed disulfide of [C14S]glutaredoxin and glutathione were obtained from the sequence-specific NMR assignments.
Suppression of isotope scrambling in cell-free protein synthesis by broadband inhibition of PLP enymes for selective 15N-labelling and production of perdeuterated proteins in H2O
Suppression of isotope scrambling in cell-free protein synthesis by broadband inhibition of PLP enymes for selective 15N-labelling and production of perdeuterated proteins in H2O
Abstract Selectively isotope labelled protein samples can be prepared in vivo or in vitro from selectively labelled amino acids but, in many cases, metabolic conversions between different amino acids result in isotope scrambling. The best results are obtained by cell-free protein synthesis, where metabolic enzymes are generally less active, but isotope scrambling can never be suppressed completely. We show that...
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02-16-2011 09:34 PM
Cell-free expression and stable isotope labelling strategies for membrane proteins
Cell-free expression and stable isotope labelling strategies for membrane proteins
Abstract Membrane proteins are highly underrepresented in the structural data-base and remain one of the most challenging targets for functional and structural elucidation. Their roles in transport and cellular communication, furthermore, often make over-expression toxic to their host, and their hydrophobicity and structural complexity make isolation and reconstitution a complicated task, especially in cases where proteins are targeted to inclusion bodies. The development of cell-free expression systems...
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01-09-2011 12:46 PM
[NMR paper] Amino acid type selective isotope labelling of the multidrug ABC transporter LmrA for
Amino acid type selective isotope labelling of the multidrug ABC transporter LmrA for solid-state NMR studies.
Related Articles Amino acid type selective isotope labelling of the multidrug ABC transporter LmrA for solid-state NMR studies.
FEBS Lett. 2004 Jun 18;568(1-3):117-21
Authors: Mason AJ, Siarheyeva A, Haase W, Lorch M, van Veen H, Glaubitz C
The ABC transporter LmrA in Lactococcus lactis confers resistance to a wide range of antibiotics and cytotoxic drugs and is a functional homologue of P-glycoprotein. Recently, solid-state NMR...
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11-24-2010 09:51 PM
Optimizing 19F NMR protein spectroscopy by fractional biosynthetic labeling
Abstract In protein NMR experiments which employ nonnative labeling, incomplete enrichment is often associated with inhomogeneous line broadening due to the presence of multiple labeled species. We investigate the merits of fractional enrichment strategies using a monofluorinated phenylalanine species, where resolution is dramatically improved over that achieved by complete enrichment. In NMR studies of calmodulin, a 148 residue calcium binding protein, 19F and 1H-15N HSQC spectra reveal a significant extent of line broadening and the appearance of minor conformers in the presence of...
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08-25-2010 03:51 PM
Optimizing (19)F NMR protein spectroscopy by fractional biosynthetic labeling.
Optimizing (19)F NMR protein spectroscopy by fractional biosynthetic labeling.
Related Articles Optimizing (19)F NMR protein spectroscopy by fractional biosynthetic labeling.
J Biomol NMR. 2010 Aug 24;
Authors: Kitevski-Leblanc JL, Evanics F, Scott Prosser R
In protein NMR experiments which employ nonnative labeling, incomplete enrichment is often associated with inhomogeneous line broadening due to the presence of multiple labeled species. We investigate the merits of fractional enrichment strategies using a monofluorinated phenylalanine species,...
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08-25-2010 02:04 PM
[NMR paper] A 1H-15N NMR study of human c-Ha-ras protein: biosynthetic incorporation of 15N-label
A 1H-15N NMR study of human c-Ha-ras protein: biosynthetic incorporation of 15N-labeled amino acids.
Related Articles A 1H-15N NMR study of human c-Ha-ras protein: biosynthetic incorporation of 15N-labeled amino acids.
J Biomol NMR. 1992 Jan;2(1):71-82
Authors: Yamasaki K, Muto Y, Ito Y, Wälchli M, Miyazawa T, Nishimura S, Yokoyama S
A 1H-15N NMR study was performed on the GDP-bound form of a truncated human c-Ha-ras oncogene product (171 amino acid residues). Resonance cross peaks of the backbone amide 1H-15N nuclei of a uniformly...
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08-21-2010 11:41 PM
[NMR paper] NMR structure of Escherichia coli glutaredoxin 3-glutathione mixed disulfide complex:
NMR structure of Escherichia coli glutaredoxin 3-glutathione mixed disulfide complex: implications for the enzymatic mechanism.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--linkinghub.elsevier.com-ihub-images-PubMedLink.gif Related Articles NMR structure of Escherichia coli glutaredoxin 3-glutathione mixed disulfide complex: implications for the enzymatic mechanism.
J Mol Biol. 1999 Feb 19;286(2):541-52
Authors: Nordstrand K, slund F, Holmgren A, Otting G, Berndt KD
Glutaredoxins (Grxs) catalyze reversible oxidation/reduction of...