One-step amino acid selective isotope labeling of proteins in prototrophic Escherichia coli strains

O’Grady, Christopher; Rempel, Benjamin L.; Sokaribo, Akosiererem S.; Nokhrin, Sergiy; Dmitriev, Oleg Y.

doi:10.1016/j.ab.2012.04.019

Cited by 20 publications

(17 citation statements)

References 16 publications

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“…After induction with 1 mM isopropyl-β-D-thiogalactoside (Roth), cells were grown for 12 h at 30 °C. For 15 N/ 13 C methionine-selective isotope-enriched samples, M9 medium contained natural abundance of NH 4 Cl, D-glucose and 0.3 mM of 15 N/ 13 C-enriched L-methionine (Isotec, 608106) 62 . α-Syn was purified using a standard purification protocol that includes a 20-min boiling step of the bacterial lysate and selective protein precipitation with ammonium sulfate (360 mg ml −1 ) 34 63 .…”

Section: Methodsmentioning

confidence: 99%

Intracellular repair of oxidation-damaged α-synuclein fails to target C-terminal modification sites

et al. 2016

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Cellular oxidative stress serves as a common denominator in many neurodegenerative disorders, including Parkinson's disease. Here we use in-cell NMR spectroscopy to study the fate of the oxidation-damaged Parkinson's disease protein alpha-synuclein (α-Syn) in non-neuronal and neuronal mammalian cells. Specifically, we deliver methionine-oxidized, isotope-enriched α-Syn into cultured cells and follow intracellular protein repair by endogenous enzymes at atomic resolution. We show that N-terminal α-Syn methionines Met1 and Met5 are processed in a stepwise manner, with Met5 being exclusively repaired before Met1. By contrast, C-terminal methionines Met116 and Met127 remain oxidized and are not targeted by cellular enzymes. In turn, persisting oxidative damage in the C-terminus of α-Syn diminishes phosphorylation of Tyr125 by Fyn kinase, which ablates the necessary priming event for Ser129 modification by CK1. These results establish that oxidative stress can lead to the accumulation of chemically and functionally altered α-Syn in cells.

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Section: Methodsmentioning

confidence: 99%

Intracellular repair of oxidation-damaged α-synuclein fails to target C-terminal modification sites

et al. 2016

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“…These procedures sometimes lack the selectivity, necessary to study large protein complexes, or suffer from poor incorporation yields. Labeled histidine, prepared by organic synthesis, has been used in the scope of the SAIL‐labeling approach for cell‐free protein synthesis, albeit with considerable economic efforts …”

Section: Methodsmentioning

confidence: 99%

Highly Selective Stable Isotope Labeling of Histidine Residues by Using a Novel Precursor in E. coli‐Based Overexpression Systems

et al. 2017

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The importance of NMR spectroscopy in unraveling the structural and dynamic properties of proteins is ever-expanding owing to progress in experimental techniques, hardware development, and novel labeling approaches. Multiple sophisticated methods of aliphatic residue labeling can be found in the literature, whereas the selective incorporation of NMR active isotopes into other amino acids still holds the potential for improvement. In order to close this methodological gap, we present a novel metabolic precursor for cell-based protein overexpression to assemble C/ H isotope patterns in the peptide backbone, as well as in side chain positions of a mechanistically distinguished histidine residue.

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“…Thus, the amino-acid selective labeling in vivo combined with modern NMR techniques is effectively employed as an essential tool for resolving resonance assignments in the crowd region. Recently, advanced methods for an efficient and simple isotope labeling were also developed using the common prototrophic E coli strains 26,27 …”

Section: Selective Isotope Labelingmentioning

confidence: 99%

A simple guide to the structural study on membrane proteins in detergents using solution NMR

Sim¹,

Lee²,

Seo³

et al. 2015

Journal of the Korean Magnetic Resonance Society

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NMR-based structural studies on membrane proteins are appreciated quite challenging due to various reasons, generally including the narrow dispersion of NMR spectra, the severe peak broadening, and the lack of long range NOEs. In spite of the poor biophysical properties, structural studies on membrane proteins have got to go on, considering their functional importance in biological systems. In this review, we provide a simple overview of the techniques generally used in structural studies of membrane proteins by solution NMR, with experimental examples of a helical membrane protein, caveolin 3. Detergent screening is usually employed as the first step and the selection of appropriate detergent is the most important for successful approach to membrane proteins. Various tools can then be applied as specialized NMR techniques in solution that include sample deteuration, amino-acid selective isotope labeling, residual dipolar coupling, and paramagnetic relaxation enhancement.

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One-step amino acid selective isotope labeling of proteins in prototrophic Escherichia coli strains

Cited by 20 publications

References 16 publications

Intracellular repair of oxidation-damaged α-synuclein fails to target C-terminal modification sites

Intracellular repair of oxidation-damaged α-synuclein fails to target C-terminal modification sites

Highly Selective Stable Isotope Labeling of Histidine Residues by Using a Novel Precursor in E. coli‐Based Overexpression Systems

A simple guide to the structural study on membrane proteins in detergents using solution NMR

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