[NMR paper] Integration of Cell-Free Expression and Solid-State NMR to Investigate the Dynamic Properties of Different Sites of the Growth Hormone Secretagogue Receptor.
Integration of Cell-Free Expression and Solid-State NMR to Investigate the Dynamic Properties of Different Sites of the Growth Hormone Secretagogue Receptor.
Related ArticlesIntegration of Cell-Free Expression and Solid-State NMR to Investigate the Dynamic Properties of Different Sites of the Growth Hormone Secretagogue Receptor.
Front Pharmacol. 2020;11:562113
Authors: Pacull EM, Sendker F, Bernhard F, Scheidt HA, Schmidt P, Huster D, Krug U
Abstract
Cell-free expression represents an attractive method to produce large quantities of selectively labeled protein for NMR applications. Here, cell-free expression was used to label specific regions of the growth hormone secretagogue receptor (GHSR) with NMR-active isotopes. The GHSR is a member of the class A family of G protein-coupled receptors. A cell-free expression system was established to produce the GHSR in the precipitated form. The solubilized receptor was refolded in vitro and reconstituted into DMPC lipid membranes. Methionines, arginines, and histidines were chosen for 13C-labeling as they are representative for the transmembrane domains, the loops and flanking regions of the transmembrane ?-helices, and the C-terminus of the receptor, respectively. The dynamics of the isotopically labeled residues was characterized by solid-state NMR measuring motionally averaged 1H-13C dipolar couplings, which were converted into molecular order parameters. Separated local field DIPSHIFT experiments under magic-angle spinning conditions using either varying cross polarization contact times or direct excitation provided order parameters for these residues showing that the C-terminus was the segment with the highest motional amplitude. The loop regions and helix ends as well as the transmembrane regions of the GHSR represent relatively rigid segments in the overall very flexible receptor molecule. Although no site resolution could be achieved in the experiments, the previously reported highly dynamic character of the receptor concluded from uniformly 13C labeled receptor samples could be further specified by this segmental labeling approach, leading to a more diversified understanding of the receptor dynamics under equilibrium conditions.
[NMR paper] Towards structural studies of self-assembled subviral particles: combining cell-free expression with 100 kHz MAS NMR.
Towards structural studies of self-assembled subviral particles: combining cell-free expression with 100 kHz MAS NMR.
Related Articles Towards structural studies of self-assembled subviral particles: combining cell-free expression with 100 kHz MAS NMR.
Angew Chem Int Ed Engl. 2018 Feb 19;:
Authors: David G, Fogeron ML, Schledorn M, Montserret R, Haselmann U, Penzel S, Badillo A, Lecoq L, André P, Nassal M, Bartenschlager R, Meier BH, Böckmann A
Abstract
Viral membrane proteins are prime targets in the combat against infection....
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02-21-2018 12:45 AM
Dynamic Nuclear Polarization/Solid-State NMR Spectroscopy of Membrane Polypeptides: Free-Radical Optimization for Matrix-Free Lipid Bilayer Samples #DNPNMR
From The DNP-NMR Blog:
Dynamic Nuclear Polarization/Solid-State NMR Spectroscopy of Membrane Polypeptides: Free-Radical Optimization for Matrix-Free Lipid Bilayer Samples #DNPNMR
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Salnikov, E.S., et al., Dynamic Nuclear Polarization/Solid-State NMR Spectroscopy of Membrane Polypeptides: Free-Radical Optimization for Matrix-Free Lipid Bilayer Samples. ChemPhysChem, 2017. 18(15): p. 2103-2113.
https://www.ncbi.nlm.nih.gov/pubmed/28574169
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09-25-2017 08:42 PM
[NMR paper] Dynamic nuclear polarization / solid-state NMR of membrane polypeptides. Free radical optimization for matrix-free lipid bilayer samples.
Dynamic nuclear polarization / solid-state NMR of membrane polypeptides. Free radical optimization for matrix-free lipid bilayer samples.
Related Articles Dynamic nuclear polarization / solid-state NMR of membrane polypeptides. Free radical optimization for matrix-free lipid bilayer samples.
Chemphyschem. 2017 Jun 02;:
Authors: Ouari O, Salnikov ES, Abel S, Karthikeyan G, Karoui H, Aussenac F, Tordo P, Bechinger B
Abstract
Dynamic Nuclear Polarization boosts the sensitivity of NMR spectroscopy by orders of magnitude making...
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06-03-2017 11:49 AM
[NMR paper] Expression, Functional Characterization, and Solid-State NMR Investigation of the G Protein-Coupled GHS Receptor in Bilayer Membranes.
Expression, Functional Characterization, and Solid-State NMR Investigation of the G Protein-Coupled GHS Receptor in Bilayer Membranes.
Expression, Functional Characterization, and Solid-State NMR Investigation of the G Protein-Coupled GHS Receptor in Bilayer Membranes.
Sci Rep. 2017 Apr 07;7:46128
Authors: Schrottke S, Kaiser A, Vortmeier G, Els-Heindl S, Worm D, Bosse M, Schmidt P, Scheidt HA, Beck-Sickinger AG, Huster D
Abstract
The expression, functional reconstitution and first NMR characterization of the human growth...
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04-08-2017 10:57 AM
High-yield Escherichia coli-based cell-free expression of human proteins
High-yield Escherichia coli-based cell-free expression of human proteins
Abstract Production of sufficient amounts of human proteins is a frequent bottleneck in structural biology. Here we describe an Escherichia coli-based cell-free system which yields mg-quantities of human proteins in N-terminal fusion constructs with the GB1 domain, which show significantly increased translation efficiency. A newly generated E. coli BL21 (DE3) RIPL-Star strain was used, which contains a variant RNase E with reduced activity and an excess of rare-codon tRNAs, and is devoid of lon and ompT protease...
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03-17-2012 07:32 AM
Cell-free expression and stable isotope labelling strategies for membrane proteins
Cell-free expression and stable isotope labelling strategies for membrane proteins
Abstract Membrane proteins are highly underrepresented in the structural data-base and remain one of the most challenging targets for functional and structural elucidation. Their roles in transport and cellular communication, furthermore, often make over-expression toxic to their host, and their hydrophobicity and structural complexity make isolation and reconstitution a complicated task, especially in cases where proteins are targeted to inclusion bodies. The development of cell-free expression systems...
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01-09-2011 12:46 PM
[NMR paper] Quantitation of protein expression in a cell-free system: Efficient detection of yiel
Quantitation of protein expression in a cell-free system: Efficient detection of yields and 19F NMR to identify folded protein.
Related Articles Quantitation of protein expression in a cell-free system: Efficient detection of yields and 19F NMR to identify folded protein.
J Biomol NMR. 2005 Jan;31(1):11-9
Authors: Neerathilingam M, Greene LH, Colebrooke SA, Campbell ID, Staunton D
We have developed an efficient and novel filter assay method, involving radioactive labelling and imaging, to quantify the expression of soluble proteins from a...
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11-24-2010 11:14 PM
Cell-free expression and labeling strategies for a new decade in solid-state NMR.
Cell-free expression and labeling strategies for a new decade in solid-state NMR.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--linkinghub.elsevier.com-ihub-images-PubMedLink.gif Related Articles Cell-free expression and labeling strategies for a new decade in solid-state NMR.
N Biotechnol. 2010 Aug 3;
Authors: Abdine A, Verhoeven MA, Warschawski DE
Although solid-state NMR and cell-free expression have recently become standard methods in biology, the combination of the two is still at a very early stage of development. In this...
Integration of Cell-Free Expression and Solid-State NMR to Investigate the Dynamic Properties of Different Sites of the Growth Hormone Secretagogue Receptor.
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