The use of Escherichia coli for recombinant protein production is a cornerstone in structural biology, particularly for nuclear magnetic resonance (NMR) spectroscopy studies. Understanding the metabolic behavior of E. coli under different carbon sources is critical for optimizing isotope labeling strategies, which are essential for protein structure determination by NMR. Recent advancements, such as mixed pyruvate labeling, have enabled improved backbone resonance assignment in large proteins, making selective isotopic labeling strategies more important than ever for NMR studies. In this study, we aimed to investigate the metabolic adaptations of E. coli when grown on pyruvate as the sole carbon source, a common condition used to achieve selective labeling for NMR spectroscopy. Using NMR-based metabolomics, we tracked key metabolic shifts throughout the culture process to better understand how pyruvate metabolism affects protein production and isotopic labeling. Our results reveal that pyruvate is rapidly depleted before IPTG induction, while acetate and lactate accumulate due to overflow metabolism. These byproducts persist after induction, indicating that pyruvate is diverted into waste pathways, which limits its efficient use in isotope incorporation. This metabolic inefficiency presents a challenge for isotopic labeling protocols that rely on pyruvate as a carbon source for NMR studies. Our results highlight the need to fine-tune pyruvate supplementation to improve metabolic efficiency and isotopic labeling, making this study directly relevant to optimizing protocols for NMR studies involving protein structure determination. These insights provide valuable guidance for enhancing the quality and yield of isotopically labeled proteins in NMR spectroscopy.
[NMR paper] Elongated Bacterial Pili as a Versatile Alignment Medium for NMR Spectroscopy
Elongated Bacterial Pili as a Versatile Alignment Medium for NMR Spectroscopy
In NMR spectroscopy, residual dipolar couplings (RDCs) have emerged as one of the most exquisite probes of biological structure and dynamics. The measurement of RDCs relies on the partial alignment of the molecule of interest, for example by using a liquid crystal as a solvent. Here, we establish bacterial type 1 pili as an alternative liquid-crystalline alignment medium for the measurement of RDCs. To achieve alignment at pilus concentrations that allow for efficient NMR sample preparation, we...
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05-31-2023 04:54 AM
Fractional enrichment of proteins using [2- 13 C]-glycerol as the carbon source facilitates measurement of excited state 13 Cα chemical shifts with improved sensitivity
Fractional enrichment of proteins using -glycerol as the carbon source facilitates measurement of excited state 13 Cα chemical shifts with improved sensitivity
Abstract
A selective isotope labeling scheme based on the utilization of -glycerol as the carbon source during protein overexpression has been evaluated for the measurement of excited state 13Cα chemical shifts using Carrâ??Purcellâ??Meiboomâ??Gill (CPMG) relaxation dispersion (RD) experiments. As expected, the fractional incorporation of label at the Cα positions is increased two-fold...
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05-20-2015 10:27 AM
Interactionsof Bacterial Cell Division Protein FtsZwith C8-Substituted Guanine Nucleotide Inhibitors. A Combined NMR,Biochemical and Molecular Modeling Perspective
Interactionsof Bacterial Cell Division Protein FtsZwith C8-Substituted Guanine Nucleotide Inhibitors. A Combined NMR,Biochemical and Molecular Modeling Perspective
Filipa Marcelo, Sonia Huecas, Laura B. Ruiz-A?vila, F. Javier Can?ada, Almudena Perona, Ana Poveda, Sonsoles Marti?n-Santamari?a, Antonio Morreale, Jesu?s Jime?nez-Barbero and Jose? M. Andreu
http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jacsat/0/jacsat.ahead-of-print/ja405515r/aop/images/medium/ja-2013-05515r_0010.gif
Journal of the American Chemical Society
DOI: 10.1021/ja405515r...
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[NMR paper] Interactions of bacterial cell division protein FtsZ with C8-substituted guanine nucleotide inhibitors. A combined NMR, biochemical and molecular modeling perspective.
Interactions of bacterial cell division protein FtsZ with C8-substituted guanine nucleotide inhibitors. A combined NMR, biochemical and molecular modeling perspective.
Interactions of bacterial cell division protein FtsZ with C8-substituted guanine nucleotide inhibitors. A combined NMR, biochemical and molecular modeling perspective.
J Am Chem Soc. 2013 Sep 30;
Authors: Marcelo F, Huecas S, Ruiz-Avila LB, Cañada FJ, Perona A, Poveda A, Martin-Santamaria S, Morreale A, Jimenez-Barbero J, Andreu JM
Abstract
FtsZ is the key protein of...
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10-02-2013 11:18 AM
[NMR paper] 13C NMR studies of the binding of medium-chain fatty acids to human serum albumin.
13C NMR studies of the binding of medium-chain fatty acids to human serum albumin.
Related Articles 13C NMR studies of the binding of medium-chain fatty acids to human serum albumin.
J Lipid Res. 1994 Mar;35(3):458-67
Authors: Kenyon MA, Hamilton JA
Binding of the medium-chain fatty acids (MCFA), octanoic (OCT) and decanoic (DEC) acid, to human serum albumin (HSA) has been studied by 13C NMR spectroscopy. NMR spectra at 35 degrees C showed an apparently homogeneous binding environment (a single, narrow resonance for the 13C-enriched carboxyl...
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[NMR paper] 13C NMR studies of the binding of medium-chain fatty acids to human serum albumin.
13C NMR studies of the binding of medium-chain fatty acids to human serum albumin.
Related Articles 13C NMR studies of the binding of medium-chain fatty acids to human serum albumin.
J Lipid Res. 1994 Mar;35(3):458-67
Authors: Kenyon MA, Hamilton JA
Binding of the medium-chain fatty acids (MCFA), octanoic (OCT) and decanoic (DEC) acid, to human serum albumin (HSA) has been studied by 13C NMR spectroscopy. NMR spectra at 35 degrees C showed an apparently homogeneous binding environment (a single, narrow resonance for the 13C-enriched carboxyl...
Exploring the biochemical landscape of bacterial medium with pyruvate as the exclusive carbon source for NMR studies
Linear Mode
