The protein human carbonic anhydrase II (HCA II) has been isotopically labeled with 2H, 13C and 15N for high-resolution NMR assignment studies and pulse sequence development. To increase the sensitivity of several key 1H/13C/15N triple-resonance correlation experiments, 2H has been incorporated into HCA II in order to decrease the rates of 13C and 1HN T2 relaxation. NMR quantities of protein with essentially complete aliphatic 2H incorporation have been obtained by growth of E. coli in defined media containing D2O, [1,2-13C2, 99%] sodium acetate, and [15N, 99%] ammonium chloride. Complete aliphatic deuterium enrichment is optimal for 13C and 15N backbone NMR assignment studies, since the 13C and 1HN T2 relaxation times and, therefore, sensitivity are maximized. In addition, complete aliphatic deuteration increases both resolution and sensitivity by eliminating the differential 2H isotopic shift observed for partially deuterated CHnDm moieties.
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment
Abstract Overexpression of isotope-labeled multi-spanning eukaryotic membrane proteins for structural NMR studies is often challenging. On the one hand, difficulties with achieving proper folding, membrane insertion, and native-like post-translational modifications frequently disqualify bacterial expression systems. On the other hand, eukaryotic cell cultures can be prohibitively expensive. One of the viable alternatives,...
nmrlearner
Proteins
0
01-22-2011 03:46 AM
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment.
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment.
Uniform isotope labeling of a eukaryotic seven-transmembrane helical protein in yeast enables high-resolution solid-state NMR studies in the lipid environment.
J Biomol NMR. 2011 Jan 19;
Authors: Fan Y, Shi L, Ladizhansky V, Brown LS
Overexpression of isotope-labeled multi-spanning eukaryotic membrane proteins for structural NMR studies is often challenging. On the one hand, difficulties...
nmrlearner
Journal club
0
01-21-2011 01:22 AM
[NMR paper] High-level bacterial expression and 15N-alanine-labeling of bovine trypsin. Applicati
High-level bacterial expression and 15N-alanine-labeling of bovine trypsin. Application to the study of trypsin-inhibitor complexes and trypsinogen activation by NMR spectroscopy.
Related Articles High-level bacterial expression and 15N-alanine-labeling of bovine trypsin. Application to the study of trypsin-inhibitor complexes and trypsinogen activation by NMR spectroscopy.
Biochemistry. 2001 May 29;40(21):6275-83
Authors: Peterson FC, Gordon NC, Gettins PG
We describe here the high-level expression of bovine trypsinogen in E. coli, its...
nmrlearner
Journal club
0
11-19-2010 08:32 PM
[NMR paper] Chain-selective isotopic labeling for NMR studies of large multimeric proteins: appli
Chain-selective isotopic labeling for NMR studies of large multimeric proteins: application to hemoglobin.
Related Articles Chain-selective isotopic labeling for NMR studies of large multimeric proteins: application to hemoglobin.
Biophys J. 2000 Aug;79(2):1146-54
Authors: Simplaceanu V, Lukin JA, Fang TY, Zou M, Ho NT, Ho C
Multidimensional, multinuclear NMR has the potential to elucidate the mechanisms of allostery and cooperativity in multimeric proteins under near-physiological conditions. However, NMR studies of proteins made up of...
nmrlearner
Journal club
0
11-19-2010 08:29 PM
[NMR paper] Uniform 13C isotope labeling of proteins with sodium acetate for NMR studies: applica
Uniform 13C isotope labeling of proteins with sodium acetate for NMR studies: application to human carbonic anhydrase II.
Related Articles Uniform 13C isotope labeling of proteins with sodium acetate for NMR studies: application to human carbonic anhydrase II.
Biochemistry. 1991 May 7;30(18):4491-4
Authors: Venters RA, Calderone TL, Spicer LD, Fierke CA
Uniform double labeling of proteins for NMR studies can be prohibitively expensive, even with an efficient expression and purification scheme, due largely to the high cost of glucose. We...
nmrlearner
Journal club
0
08-21-2010 11:16 PM
Auto-induction medium containing glyphosate for high-level incorporation of ... - Bio
Auto-induction medium containing glyphosate for high-level incorporation of ... - BioTechniques.com
<img alt="" height="1" width="1" />
Auto-induction medium containing glyphosate for high-level incorporation of ...
BioTechniques.com
Quantitation of protein expression in a cell-free system: efficient detection of yields and 19 F NMR to identify folded protein. J. Biomol. NMR 31:11-19. 7. ...
Read here
nmrlearner
Online News
0
08-16-2010 03:50 AM
Site-specific labeling of nucleotides for making RNA for high resolution NMR studies
Abstract Escherichia coli (E. coli) is a versatile organism for making nucleotides labeled with stable isotopes (13C, 15N, and/or 2H) for structural and molecular dynamics characterizations. Growth of a mutant E. coli strain deficient in the pentose phosphate pathway enzyme glucose-6-phosphate dehydrogenase (K10-1516) on 2-13C-glycerol and 15N-ammonium sulfate in Studier minimal medium enables labeling at sites useful for NMR spectroscopy. However, 13C-sodium formate combined with 13C-2-glycerol in the growth media adds labels to new positions. In the absence of labeled formate, both C5 and...
nmrlearner
Journal club
0
08-14-2010 04:19 AM
High Resolution 1H Detected 1H,13C Correlation Spectra in MAS Solid-State NMR using Deuterated Proteins with Selective 1H,2H Isotopic Labeling of Methyl Groups
High Resolution <SUP>1</SUP>H Detected <SUP>1</SUP>H,<SUP>13</SUP>C Correlation Spectra in MAS Solid-State NMR using Deuterated Proteins with Selective <SUP>1</SUP>H,<SUP>2</SUP>H Isotopic Labeling of Methyl Groups
Vipin Agarwal, Anne Diehl, Nikolai Skrynnikov, and Bernd Reif
J. Am. Chem. Soc.; 2006; 128(39) pp 12620 - 12621;
Abstract:
MAS solid-state NMR experiments applied to biological solids are still hampered by low sensitivity and resolution. In this work, we employ a deuteration scheme in which individual methyl groups are selectively protonated. This labeling scheme...
High-level 2H/13C/15N labeling of proteins for NMR studies.
Linear Mode
