Related ArticlesCharacterizing proteins in a native bacterial environment using solid-state NMR spectroscopy.
Nat Protoc. 2021 Jan 13;:
Authors: Narasimhan S, Pinto C, Lucini Paioni A, van der Zwan J, Folkers GE, Baldus M
Abstract
For a long time, solid-state nuclear magnetic resonance (ssNMR) has been employed to study complex biomolecular systems at the detailed chemical, structural, or dynamic level. Recent progress in high-resolution and high-sensitivity ssNMR, in combination with innovative sample preparation and labeling schemes, offers novel opportunities to study proteins in their native setting irrespective of the molecular tumbling rate. This protocol describes biochemical preparation schemes to obtain cellular samples of both soluble as well as insoluble or membrane-associated proteins in bacteria. To this end, the protocol is suitable for studying a protein of interest in both whole cells and in cell envelope or isolated membrane preparations. In the first stage of the procedure, an appropriate strain of Escherichia coli (DE3) is transformed with a plasmid of interest harboring the protein of interest under the control of an inducible T7 promoter. Next, the cells are adapted to grow in minimal (M9) medium. Before the growth enters stationary phase, protein expression is induced, and shortly thereafter, the native E. coli RNA polymerase is inhibited using rifampicin for targeted labeling of the protein of interest. The cells are harvested after expression and prepared for ssNMR rotor filling. In addition to conventional 13C/15N-detected ssNMR, we also outline how these preparations can be readily subjected to multidimensional ssNMR experiments using dynamic nuclear polarization (DNP) or proton (1H) detection schemes. We estimate that the entire preparative procedure until NMR experiments can be started takes 3-5 days.
PMID: 33442051 [PubMed - as supplied by publisher]
[NMR paper] Combined 1H-Detected Solid-State NMR Spectroscopy and Electron Cryotomography to Study Membrane Proteins across Resolutions in Native Environments.
Combined 1H-Detected Solid-State NMR Spectroscopy and Electron Cryotomography to Study Membrane Proteins across Resolutions in Native Environments.
Related Articles Combined 1H-Detected Solid-State NMR Spectroscopy and Electron Cryotomography to Study Membrane Proteins across Resolutions in Native Environments.
Structure. 2017 Dec 06;:
Authors: Baker LA, Sinnige T, Schellenberger P, de Keyzer J, Siebert CA, Driessen AJM, Baldus M, Grünewald K
Abstract
Membrane proteins remain challenging targets for structural biology,...
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12-19-2017 11:31 AM
Combined 1H-Detected Solid-State NMR Spectroscopy and Electron Cryotomography to Study Membrane Proteins across Resolutions in Native Environments
Combined 1H-Detected Solid-State NMR Spectroscopy and Electron Cryotomography to Study Membrane Proteins across Resolutions in Native Environments
Publication date: Available online 14 December 2017
Source:Structure</br>
Author(s): Lindsay A. Baker, Tessa Sinnige, Pascale Schellenberger, Jeanine de Keyzer, C. Alistair Siebert, Arnold J.M. Driessen, Marc Baldus, Kay Grünewald</br>
Membrane proteins remain challenging targets for structural biology, despite much effort, as their native environment is heterogeneous and complex. Most methods rely on...
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12-15-2017 09:07 PM
Journal Highlight: Membrane proteins in their native habitat as seen by solid-state NMR spectroscopy
Journal Highlight: Membrane proteins in their native habitat as seen by solid-state NMR spectroscopy
http://www.spectroscopynow.com/common/images/thumbnails/14fff98e0e1.jpgRecent progress in the solid-state NMR spectroscopy of membrane proteins within a cellular membrane has been reviewed, along with requirements for sample preparation.
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09-28-2015 06:31 PM
Membrane proteins in their native habitat as seen by solid-state NMR spectroscopy
Membrane proteins in their native habitat as seen by solid-state NMR spectroscopy
Abstract
Membrane proteins play many critical roles in cells, mediating flow of material and information across cell membranes. They have evolved to perform these functions in the environment of a cell membrane, whose physicochemical properties are often different from those of common cell membrane mimetics used for structure determination. As a result, membrane proteins are difficult to study by traditional methods of structural biology, and they are significantly underrepresented in the protein structure...
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05-27-2015 10:39 AM
[NMR paper] Membrane proteins in their native habitat as seen by solid-state NMR spectroscopy.
Membrane proteins in their native habitat as seen by solid-state NMR spectroscopy.
Related Articles Membrane proteins in their native habitat as seen by solid-state NMR spectroscopy.
Protein Sci. 2015 May 13;
Authors: Brown LS, Ladizhansky V
Abstract
Membrane proteins play many critical roles in cells, mediating flow of material and information across cell membranes. They have evolved to perform these functions in the environment of a cell membrane, whose physicochemical properties are often different from those of common cell...
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05-15-2015 08:02 PM
Membrane proteins in their native habitat as seen by solid-state NMR spectroscopy
Membrane proteins in their native habitat as seen by solid-state NMR spectroscopy
Abstract
Membrane proteins play many critical roles in cells, mediating flow of material and information across cell membranes. They have evolved to perform these functions in the environment of a cell membrane, whose physicochemical properties are often different from those of common cell membrane mimetics used for structure determination. As a result, membrane proteins are difficult to study by traditional methods of structural biology, and they are significantly underrepresented in the protein...
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05-13-2015 02:01 PM
[NMR paper] Structure Determination of Membrane Proteins in Their Native Phospholipid Bilayer Environment by Rotationally Aligned Solid-State NMR Spectroscopy.
Structure Determination of Membrane Proteins in Their Native Phospholipid Bilayer Environment by Rotationally Aligned Solid-State NMR Spectroscopy.
Structure Determination of Membrane Proteins in Their Native Phospholipid Bilayer Environment by Rotationally Aligned Solid-State NMR Spectroscopy.
Acc Chem Res. 2013 Jul 5;
Authors: Opella SJ
Abstract
One of the most important topics in experimental structural biology is determining the structures of membrane proteins. These structures represent one-third of all of the information...
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07-09-2013 02:47 PM
[NMR paper] Solid State NMR Strategy for Characterizing Native Membrane Protein Structures.
Solid State NMR Strategy for Characterizing Native Membrane Protein Structures.
Related Articles Solid State NMR Strategy for Characterizing Native Membrane Protein Structures.
Acc Chem Res. 2013 Mar 7;
Authors: Murray DT, Das N, Cross TA
Abstract
Unlike water soluble proteins, the structures of helicaltransmembrane proteins depend on a very complex environment. These proteins sit in the midst of dramatic electrical and chemical gradients and are often subject to variations in the lateral pressure profile, order parameters, dielectric...
Characterizing proteins in a native bacterial environment using solid-state NMR spectroscopy.
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