The 19F - 13C HMQC spectrum of trifluoroacetic acid is shown in the figure below.
The data were collected with a delay appropriate for a 19F - 13C J coupling constant between the 1JF-C coupling constant of 284 Hz and the 2JF-C coupling constant of 44 Hz. The top and side traces are the one-pulse 19F and 13C spectra, respectively. Why are the HMQC responses not at the same 19F chemical shift and why aren't they correlated to the peak in the 19F spectrum? In order to answer these questions one must take into consideration the 19F - 12, 13C isotope effects. The chemical shift of the fluorine depends on whether it is bound to a 12C or a 13C. The effect is largest across one bond and gets smaller over multiple bonds. The 19F NMR spectrum for trifluoroacetic acid is shown in the figure below with and without 13C broadband decoupling in the upper and lower traces, respectively.
Approximately 98% of the trifluoroacetic acid is the 12CF3-12COOH isotopomer, giving rise to a large singlet plotted off-scale in the figure. Approximately 1% of the signal is from the 13CF3-12COOH isotoponer giving rise to a doublet with 1JF-C = 284 Hz and aapproximately 1% of the signal is from the 12CF3-13COOH isotoponer giving rise to a doublet with 2JF-C = 44 Hz. All of these signals are clearly present in the lower trace of the figure. When 13C broadband decoupling is applied, the doublets collapse into singlets. The singlets from each of the isotopomers are resolved in the top trace. The one-bond 19F - 12, 13C isotope effect is 0.13 ppm and the two-bond effect is 0.02 ppm. The figure below shows the same HMQC data with the spectrum from the top trace used as a projection.
One can see that the HMQC responses are correlated to their respective isotopomers. These effects are also present in 1H - 13C HMQC spectra, but the 1H - 12, 13C isotope effect is much smaller than the 19F - 12, 13C isotope effect.
A simple protocol for amino acid type selective isotope labeling in insect cells with improved yields and high reproducibility
A simple protocol for amino acid type selective isotope labeling in insect cells with improved yields and high reproducibility
Abstract An easy to use and robust approach for amino acid type selective isotope labeling in insect cells is presented. It relies on inexpensive commercial media and can be implemented in laboratories without sophisticated infrastructure. In contrast to previous protocols, where either high protein amounts or high incorporation ratios were obtained, here we achieve both at the same time. By supplementing media with a well considered amount of yeast extract,...
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[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
On the measurement of 15N-{1H} nuclear Overhauser effects. 2. Effects of the saturati
On the measurement of 15N-{1H} nuclear Overhauser effects. 2. Effects of the saturation scheme and water signal suppression
Publication year: 2010
Source: Journal of Magnetic Resonance, In Press, Accepted Manuscript, Available online 24 September 2010</br>
Fabien, Ferrage , Amy, Reichel , Shibani, Battacharya , David, Cowburn , Ranajeet, Ghose</br>
Measurement of steady-state 15N-{1H} nuclear Overhauser effects forms a cornerstone of most methods to determine protein backbone dynamics from spin-relaxation data, since it is the most reliable probe of very fast motions on the ps-ns...
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09-25-2010 07:47 AM
[NMR paper] Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for
Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for NMR analysis.
Related Articles Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for NMR analysis.
J Biomol NMR. 1995 Sep;6(2):129-34
Authors: Kigawa T, Muto Y, Yokoyama S
For the application of multidimensional NMR spectroscopy to larger proteins, it would be useful to perform selective labeling of one of the 20 amino acids. For some amino acids, however, amino acid metabolism drastically reduces the efficiency and selectivity...
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A simple method for amino acid selective isotope labeling of recombinant proteins in E. coli
A simple method for amino acid selective isotope labeling of recombinant proteins in E. coli
Kit I. Tong, Masayuki Yamamoto and Toshiyuki Tanaka
Journal of Biomolecular NMR; 2008; 42(1); pp 59-67
Abstract:
A simple and user-friendly method of labeling protein selectively with amino acids in vivo is introduced. This technique does not require the use of transaminase-deficient or auxotrophic strains. By manipulating the product feedback inhibitory loops of the E. coli amino acid metabolic pathways and, if necessary, by using enzyme inhibitors, proteins were labeled efficiently in vivo...