Related ArticlesEstimation of the intracellular free ADP concentration by 19F NMR studies of fluorine-labeled yeast phosphoglycerate kinase in vivo.
Biochemistry. 1993 May 11;32(18):4895-902
Authors: Williams SP, Fulton AM, Brindle KM
Yeast phosphoglycerate kinase was selectively fluorine-labeled in vivo by inducing enzyme synthesis in stationary phase cells in the presence of 5-fluorotryptophan. Inducible expression was obtained using a galactose-inducible expression vector containing the yeast phosphoglycerate kinase coding sequence. 19F NMR measurements on intact cells showed two resolved resonances, from the two tryptophan residues in the protein, which underwent reversible changes in chemical shift under different metabolic conditions. Measurements in vitro showed that the difference in the chemical shifts of these two resonances was dependent on the adenine nucleotide concentration, in particular the MgADP concentration. A comparison of the spectra obtained in vitro with those obtained from the intact cell indicated that in glucose-fed cells the cytosolic free MgADP concentration was less than 50 microM, which is significantly lower than the concentrations measured in whole-cell extracts.
[Question from NMRWiki Q&A forum] incorporating fluorine into proteins
incorporating fluorine into proteins
Hi
I was considering the possibility of trying some fluorine NMR and was wanting to know if anyone knew any simple ways of incorporating Fluorine probes into my protein relatively cheaply or using E.coli. I would be particularly interested in using fluro-tryptophan for dual use as NMR and florescence probes. Also does anyone have a feel for the size limitations of 19F probes literature always quotes around 100KDa but is it possible to go considerably higher, say 500?
any advice would be great
Tom
nmrlearner
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02-02-2012 09:43 PM
[NMR paper] Cell-free synthesis of 15N-labeled proteins for NMR studies.
Cell-free synthesis of 15N-labeled proteins for NMR studies.
Related Articles Cell-free synthesis of 15N-labeled proteins for NMR studies.
IUBMB Life. 2005 Sep;57(9):615-22
Authors: Ozawa K, Dixon NE, Otting G
Modern cell-free in vitro protein synthesis systems present powerful tools for the synthesis of isotope-labeled proteins in high yields. The production of selectively 15 N-labeled proteins from 15 N-labeled amino acids is particularly economic and yields are often sufficient to analyze the proteins very quickly by two-dimensional NMR...
nmrlearner
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12-01-2010 06:56 PM
[NMR paper] 15N NMR relaxation studies of free and inhibitor-bound 4-oxalocrotonate tautomerase:
15N NMR relaxation studies of free and inhibitor-bound 4-oxalocrotonate tautomerase: backbone dynamics and entropy changes of an enzyme upon inhibitor binding.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--pubs.acs.org-images-acspubs.jpg Related Articles 15N NMR relaxation studies of free and inhibitor-bound 4-oxalocrotonate tautomerase: backbone dynamics and entropy changes of an enzyme upon inhibitor binding.
Biochemistry. 1996 Dec 17;35(50):16036-47
Authors: Stivers JT, Abeygunawardana C, Mildvan AS
The solution secondary...
nmrlearner
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08-22-2010 02:20 PM
[NMR paper] 19F-NMR study of the effect of lead on intracellular free calcium in human platelets.
19F-NMR study of the effect of lead on intracellular free calcium in human platelets.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--linkinghub.elsevier.com-ihub-images-PubMedLink.gif Related Articles 19F-NMR study of the effect of lead on intracellular free calcium in human platelets.
Biochim Biophys Acta. 1991 May 17;1092(3):341-6
Authors: Dowd TL, Gupta RK
Lead has been shown to affect calcium homeostasis. However, there is no prior evidence to indicate an effect of low concentrations of lead in the environment (approximately 1...
nmrlearner
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08-21-2010 11:16 PM
Cell-free protein synthesis of perdeuterated proteins for NMR studies
Cell-free protein synthesis of perdeuterated proteins for NMR studies
Touraj Etezady-Esfarjani, Sebastian Hiller, Cristina Villalba and Kurt Wüthrich
Journal of Biomolecular NMR; 2007; 39(3); pp 229-238
Abstract:
Cell-free protein synthesis protocols for uniformly deuterated proteins typically yield low, non-uniform deuteration levels. This paper introduces an E. coli cell-extract, D-S30, which enables efficient production of proteins with high deuteration levels for all non-labile hydrogen atom positions. Potential applications of the new protocol may include production of proteins...
Deano
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08-14-2008 10:01 PM
Online help for measurement concentration by NMR
Below are links to online resources that can help students to understand how to measure concentration of substances with NMR. If you know websites that discuss this issue, please post links to them in this thread. Quantitative Nuclear Magnetic Resonance, Chemistry 3113, University of North Carolina Charlotte
Chem152, Rice University
Relevant softwareACD/Combi NMR