N-terminal gluconoylation is a moderately widespread modification in recombinant proteins expressed in Escherichia coli, in particular in proteins bearing an N-terminal histidine-tag. This post-translational modification has been investigated mainly by mass spectrometry. Although its NMR signals must have been observed earlier in spectra of 13C/15N labeled proteins, their chemical shifts were not yet reported. Here we present the complete 1H and 13C chemical shift assignment of the N-terminal gluconoyl post-translational modification, based on a selection of His-tagged protein constructs (CCL2, hnRNP A1 and Lin28) starting with Met-Gly-...-(His)6. In addition, we show that the modification can hydrolyze over time, resulting in a free N-terminus and gluconate. This leads to the disappearance of the gluconoyl signals and the appearance of gluconate signals during the NMR measurements. The chemical shifts presented here can now be used as a reference for the identification of gluconoylation in recombinant proteins, in particular when isotopically labeled.
[NMR paper] Production of isotope-labeled proteins in insect cells for NMR.
Production of isotope-labeled proteins in insect cells for NMR.
Related Articles Production of isotope-labeled proteins in insect cells for NMR.
J Biomol NMR. 2018 Apr 23;:
Authors: Franke B, Opitz C, Isogai S, Grahl A, Delgado L, Gossert AD, Grzesiek S
Abstract
Baculovirus-infected insect cells have become a powerful tool to express recombinant proteins for structural and functional studies by NMR spectroscopy. This article provides an introduction into the insect cell/baculovirus expression system and its use for the production...
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04-26-2018 02:15 AM
Production of isotope-labeled proteins in insect cells for NMR
Production of isotope-labeled proteins in insect cells for NMR
Abstract
Baculovirus-infected insect cells have become a powerful tool to express recombinant proteins for structural and functional studies by NMR spectroscopy. This article provides an introduction into the insect cell/baculovirus expression system and its use for the production of recombinant isotope-labeled proteins. We discuss recent advances in inexpensive isotope-labeling methods using labeled algal or yeast extracts as the amino acid source and give examples of advanced NMR...
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04-23-2018 05:00 PM
[NMR paper] Differentially Isotope-Labeled Nucleosomes to Study Asymmetric Histone Modification Crosstalk by Time-Resolved NMR Spectroscopy.
Differentially Isotope-Labeled Nucleosomes to Study Asymmetric Histone Modification Crosstalk by Time-Resolved NMR Spectroscopy.
Related Articles Differentially Isotope-Labeled Nucleosomes to Study Asymmetric Histone Modification Crosstalk by Time-Resolved NMR Spectroscopy.
Angew Chem Int Ed Engl. 2016 May 24;
Authors: Liokatis S, Klingberg R, Tan S, Schwarzer D
Abstract
Post-translational modifications (PTMs) of histones regulate chromatin structure and function. Because nucleosomes contain two copies each of the four core...
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05-25-2016 02:33 PM
CIL partners to provide more isotope-labeled proteins to pharma - OutSourcing-Pharma.com
CIL partners to provide more isotope-labeled proteins to pharma - OutSourcing-Pharma.com
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CIL partners to provide more isotope-labeled proteins to pharma
OutSourcing-Pharma.com
"Nexomics success in developing techniques to determine protein structure using Nuclear Magnetic Resonance (NMR) spectroscopy nicely complements CIL's mission to offer isotope enriched reagents used to produce labeled recombinant protein.
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01-30-2014 05:38 PM
A strong 13C chemical shift signature provides the coordination mode of histidines in zinc-binding proteins
A strong 13C chemical shift signature provides the coordination mode of histidines in zinc-binding proteins
Abstract Zinc is the second most abundant metal ion incorporated in proteins, and is in many cases a crucial component of protein three-dimensional structures. Zinc ions are frequently coordinated by cysteine and histidine residues. Whereas cysteines bind to zinc via their unique Sγ atom, histidines can coordinate zinc with two different coordination modes, either Nδ1 or Nε2 is coordinating the zinc ion. The determination of this coordination mode is crucial for the accurate...
Influence of substrate modification and C-terminal truncation on the active site structure of substrate-bound heme oxygenase from Neisseriae meningitidis; A 1H NMR study.
Influence of substrate modification and C-terminal truncation on the active site structure of substrate-bound heme oxygenase from Neisseriae meningitidis; A 1H NMR study.
Influence of substrate modification and C-terminal truncation on the active site structure of substrate-bound heme oxygenase from Neisseriae meningitidis; A 1H NMR study.
Biochemistry. 2011 Aug 27;
Authors: Peng D, Satterlee JD, Ma LH, Dallas JL, Smith KM, Zhang X, Sato M, La Mar GN
Abstract
Heme oxygenase, HO, from the pathogenic bacterium N. meningitidis, NmHO, which...
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08-30-2011 04:52 PM
An economical method for producing stable-isotope labeled proteins by the E. coli cel
An economical method for producing stable-isotope labeled proteins by the E. coli cell-free system
Abstract Improvement of the cell-free protein synthesis system (CF) over the past decade have made it one of the most powerful protein production methods. The CF approach is especially useful for stable-isotope (SI) labeling of proteins for NMR analysis. However, it is less popular than expected, partly because the SI-labeled amino acids used for SI labeling by the CF are too expensive. In the present study, we developed a simple and inexpensive method for producing an SI-labeled protein...
The NMR signature of gluconoylation: a frequent N-terminal modification of isotope-labeled proteins
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