Abstract
NMR spectroscopy is a method of choice to characterize structure, function, and dynamics of integral membrane proteins at atomic resolution. Here, we describe protocols for sample preparation and characterization by NMR spectroscopy of two integral membrane proteins with different architecture, the ?-helical membrane protein MsbA and the ?-barrel membrane protein BamA. The protocols describe recombinant expression in E. coli, protein refolding, purification, and reconstitution in suitable membrane mimetics, as well as key setup steps for basic NMR experiments. These include experiments on protein samples in the solid state under magic angle spinning (MAS) conditions and experiments on protein samples in aqueous solution. Since MsbA and BamA are typical examples of their respective architectural classes, the protocols presented here can*also serve as*a reference for other integral membrane proteins.
[U. of Ottawa NMR Facility Blog] Glycine as a 13C CPMAS Setup Sample
Glycine as a 13C CPMAS Setup Sample
Glycine is an excellent setup compound for 13C CPMAS NMR measurements. Its utility in this regard has been described in detail.1,2 It can easily be observed in one scan and has reasonably short 1H T1's, allowing it to be used for 1H 90° pulse calibration and to setup the Hartmann Hahn matching condition. The width of the methylene carbon signal can be conveniently used to evaluate the proton decoupling efficiency. The width and shape of the carbonyl signal are very sensitive to the angle at which the sample is spun and can be used to set the magic...
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06-21-2018 10:11 PM
[NMR paper] Macrodiscs Comprising SMALPs for Oriented Sample Solid-State NMR Spectroscopy of Membrane Proteins.
Macrodiscs Comprising SMALPs for Oriented Sample Solid-State NMR Spectroscopy of Membrane Proteins.
Related Articles Macrodiscs Comprising SMALPs for Oriented Sample Solid-State NMR Spectroscopy of Membrane Proteins.
Biophys J. 2018 Jun 15;:
Authors: Radoicic J, Park SH, Opella SJ
Abstract
Macrodiscs, which are magnetically alignable lipid bilayer discs with diameters of >30*nm, were obtained by solubilizing protein-containing liposomes with styrene-maleic acid copolymers. Macrodiscs provide a detergent-free phospholipid bilayer...
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06-20-2018 08:56 PM
Macrodiscs Comprising SMALPs for Oriented Sample Solid-State NMR Spectroscopy of Membrane Proteins
Macrodiscs Comprising SMALPs for Oriented Sample Solid-State NMR Spectroscopy of Membrane Proteins
Publication date: Available online 15 June 2018
Source:Biophysical Journal</br>
Author(s): Jasmina Radoicic, Sang Ho Park, Stanley J. Opella</br>
Macrodiscs, which are magnetically alignable lipid bilayer discs with diameters of >30*nm, were obtained by solubilizing protein-containing liposomes with styrene-maleic acid copolymers. Macrodiscs provide a detergent-free phospholipid bilayer environment for biophysical and functional studies of membrane proteins under...
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06-16-2018 07:20 AM
[NMR paper] Sample Preparation for Membrane Protein Structural Studies by Solid-State NMR.
Sample Preparation for Membrane Protein Structural Studies by Solid-State NMR.
Related Articles Sample Preparation for Membrane Protein Structural Studies by Solid-State NMR.
Methods Mol Biol. 2017;1635:345-358
Authors: Lacabanne D, Kunert B, Gardiennet C, Meier BH, Bo Ckmann A
Abstract
Conformational studies of membrane proteins remain a challenge in the field of structural biology, and in particular the investigation of the proteins in a native-like lipid environment. Solid-state NMR presents a valuable opportunity for...
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07-30-2017 08:04 PM
Efficient DNP NMR of membrane proteins: sample preparation protocols, sensitivity, and radical location
From The DNP-NMR Blog:
Efficient DNP NMR of membrane proteins: sample preparation protocols, sensitivity, and radical location
Liao, S.Y., et al., Efficient DNP NMR of membrane proteins: sample preparation protocols, sensitivity, and radical location. J Biomol NMR, 2016: p. 1-15.
http://www.ncbi.nlm.nih.gov/pubmed/26873390
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02-25-2016 05:21 AM
[NMR paper] Efficient DNP NMR of membrane proteins: sample preparation protocols, sensitivity, and radical location.
Efficient DNP NMR of membrane proteins: sample preparation protocols, sensitivity, and radical location.
Related Articles Efficient DNP NMR of membrane proteins: sample preparation protocols, sensitivity, and radical location.
J Biomol NMR. 2016 Feb 12;
Authors: Liao SY, Lee M, Wang T, Sergeyev IV, Hong M
Abstract
Although dynamic nuclear polarization (DNP) has dramatically enhanced solid-state NMR spectral sensitivities of many synthetic materials and some biological macromolecules, recent studies of membrane-protein DNP using...
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02-14-2016 08:25 PM
Efficient DNP NMR of membrane proteins: sample preparation protocols, sensitivity, and radical location
Efficient DNP NMR of membrane proteins: sample preparation protocols, sensitivity, and radical location
Abstract
Although dynamic nuclear polarization (DNP) has dramatically enhanced solid-state NMR spectral sensitivities of many synthetic materials and some biological macromolecules, recent studies of membrane-protein DNP using exogenously doped paramagnetic radicals as polarizing agents have reported varied and sometimes surprisingly limited enhancement factors. This motivated us to carry out a systematic evaluation of sample preparation protocols...
Sample Preparation and Technical Setup for NMR Spectroscopy with Integral Membrane Proteins.
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