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Production of isotope-labeled proteins in insect cells for NMR
Apr 23, 2018 - 5:00 PM - by nmrlearner
nmrlearner's Avatar 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 applications for proteins, which have become accessible by this eukaryotic expression host.



Source: Journal of Biomolecular NMR
0 Replies | 15 Views
Real-time monitoring of enzyme-assisted animal protein hydrolysis by NMR spectroscopy – An NMR reactomics concept
Apr 23, 2018 - 5:00 PM - by nmrlearner
nmrlearner's Avatar Real-time monitoring of enzyme-assisted animal protein hydrolysis by NMR spectroscopy – An NMR reactomics concept

Publication date: Available online 23 April 2018
Source:LWT

Author(s): Ulrik K. Sundekilde, Lise Jarno, Nina Eggers, Hanne Christine Bertram

This study presents an application of proton (1H) NMR spectroscopy for monitoring of enzyme-assisted hydrolysis of muscle protein under both at-line and real-time conditions. Measurements were carried out on sample material >1 g, and two different enzymes (alcalase and papain) were examined. The real-time monitoring of reactions was carried out directly in the NMR spectrometer, and the technique enabled the dynamic and quantitative detection of a total of 40 metabolites. The approach enabled to follow time evolution and kinetic modelling allowed extraction of kinetic parameters; a was calculated to 0.051 min-1 and 0.029 min-1 for alcalase and papain, respectively, revealing a higher hydrolysis velocity for alcalase than papain. The study demonstrates that NMR spectroscopy is an excellent analytical platform for real-time monitoring of the major metabolite composition changes occurring during an enzymatic hydrolysis. Industrial relevance: Protein hydrolysis is a commonly applied technology in food industry and the presented NMR methodology for kinetic studies of metabolite generation during a hydrolysis process would be applicable for testing and optimization of different raw materials... [Read More]
0 Replies | 5 Views
Magnetic-Field-Dependent Lifetimes of Hyperpolarized 13C Spins at Cryogenic Temperature
Apr 23, 2018 - 5:00 PM - by nmrlearner
nmrlearner's Avatar From The DNP-NMR Blog:

Magnetic-Field-Dependent Lifetimes of Hyperpolarized 13C Spins at Cryogenic Temperature

Niedbalski, P., et al., Magnetic-Field-Dependent Lifetimes of Hyperpolarized 13C Spins at Cryogenic Temperature. The Journal of Physical Chemistry B, 2018. 122(6): p. 1898-1904.


https://doi.org/10.1021/acs.jpcb.8b00630


Using a home-built cryogen-free dynamic nuclear polarization (DNP) system with a variable magnetic field capability, 13C spin–lattice T1 relaxation times of hyperpolarized [1-13C] carboxylates (sodium acetate, glycine, sodium pyruvate, and pyruvic acid) doped with trityl OX063 free radical were systematically measured for the first time at different field strengths up to 9 T at T = 1.8 K. Our data reveal that the 13C T1 values of these frozen hyperpolarized 13C samples vary drastically with the applied magnetic field B according to an apparent empirical power-law dependence (13C T1 ? B?, 2.3 < ? < 3.1), with relaxation values ranging from a few hundred seconds at 1 T to over 200,000 s at fields close to 9 T. This low temperature relaxation behavior can be ascribed approximately to a model that accounts for the combined effect of 13C–1H intramolecular dipolar interaction and the relaxation contribution from the paramagnetic impurities present in the DNP sample. Since the lifetime or T1 storage of the hyperpolarized state is intimately linked to DNP efficiency, these 13C relaxation data at cryogenic temperature have important theoretical and experimental implications as the DNP of 13C-labeled biomolecules is pushed to higher magnetic fields.

... [Read More]
0 Replies | 9 Views
[NMR paper] Protein-Solvent Interfaces in Human Y145Stop Prion Protein Amyloid Fibrils Probed by Paramagnetic Solid-State NMR Spectroscopy.
Apr 22, 2018 - 10:46 PM - by nmrlearner
nmrlearner's Avatar Protein-Solvent Interfaces in Human Y145Stop Prion Protein Amyloid Fibrils Probed by Paramagnetic Solid-State NMR Spectroscopy.

Related Articles Protein-Solvent Interfaces in Human Y145Stop Prion Protein Amyloid Fibrils Probed by Paramagnetic Solid-State NMR Spectroscopy.

J Struct Biol. 2018 Apr 18;:

Authors: Aucoin D, Xia Y, Theint T, Nadaud PS, Surewicz K, Surewicz WK, Jaroniec CP

Abstract
The C-terminally truncated Y145Stop variant of prion protein (PrP23-144), which is associated with heritable PrP cerebral amyloid angiopathy in humans and also capable of triggering a transmissible prion disease in mice, serves as a useful in vitro model for investigating the molecular and structural basis of amyloid strains and cross-seeding specificities. Here, we determine the protein-solvent interfaces in human PrP23-144 amyloid fibrils generated from recombinant 13C,15N-enriched protein and incubated in aqueous solution containing paramagnetic Cu(II)-EDTA, by measuring residue-specific 15N longitudinal paramagnetic relaxation enhancements using two-dimensional magic-angle spinning solid-state NMR spectroscopy. To further probe the interactions of the amyloid core residues with solvent molecules we perform complementary measurements of amide hydrogen/deuterium exchange detected by solid-state NMR and solution NMR methods. The solvent accessibility data are evaluated in the context of the structural... [Read More]
0 Replies | 17 Views
[NMR paper] Anti-cancer effects of emodin on HepG2 cells as revealed by 1H-NMR based metabolic profiling.
Apr 21, 2018 - 10:14 AM - by nmrlearner
nmrlearner's Avatar Anti-cancer effects of emodin on HepG2 cells as revealed by 1H-NMR based metabolic profiling.

Related Articles Anti-cancer effects of emodin on HepG2 cells as revealed by 1H-NMR based metabolic profiling.

J Proteome Res. 2018 Apr 20;:

Authors: Xing YX, Li MH, Tao L, Ruan LY, Hong W, Chen C, Zhao WL, Xu H, Chen JF, Wang JS

Abstract
Hepatic carcinoma is one of the most common cancers in the world with a high incidence. Emodin is an anthraquinone derived from Polygonum multiflorum Thunb, possessing anti-cancer activity. The purpose of this study is to investigate the anti-cancer effect of different dosages of emodin on HepG2 cells using 1H-NMR based metabolic approach complemented with qRT-PCR and flow cytometry to identify potential markers and discover the targets to explore the underlying mechanism. Emodin can dose-dependently inhibit the growth of HepG2 cells, perturb cell cycle progression, down-regulate the expression of genes and proteins related to glycolysis and trigger intracellular ROS generation. Orthogonal signal correction partial least-squares discriminant analysis (OSC-PLS-DA) and correlation network analysis of the 1H NMR data showed significant changes in many endogenous metabolites after emodin exposure concerning oxidative stress and disturbances in amino acid and energy metabolism. These findings are helpful to understand the anti-cancer mechanism of emodin and provide a... [Read More]
0 Replies | 27 Views
[NMR paper] Solution NMR Studies of Anesthetic Interactions with Ion Channels.
Apr 21, 2018 - 10:14 AM - by nmrlearner
nmrlearner's Avatar Solution NMR Studies of Anesthetic Interactions with Ion Channels.

Related Articles Solution NMR Studies of Anesthetic Interactions with Ion Channels.

Methods Enzymol. 2018;603:49-66

Authors: Bondarenko V, Wells M, Xu Y, Tang P

Abstract
NMR spectroscopy is one of the major tools to provide atomic resolution protein structural information. It has been used to elucidate the molecular details of interactions between anesthetics and ion channels, to identify anesthetic binding sites, and to characterize channel dynamics and changes introduced by anesthetics. In this chapter, we present solution NMR methods essential for investigating interactions between ion channels and general anesthetics, including both volatile and intravenous anesthetics. Case studies are provided with a focus on pentameric ligand-gated ion channels and the voltage-gated sodium channel NaChBac.


PMID: 29673534 [PubMed - in process]



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