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  Backbone assignment of perdeuterated proteins by solid-state NMR using proton detection and ultrafast magic-angle spinning.

Fricke, P., Chevelkov, V., Zinke, M., Giller, K., Becker, S., & Lange, A. (2017). Backbone assignment of perdeuterated proteins by solid-state NMR using proton detection and ultrafast magic-angle spinning. Nature Protocols, 12(4), 764-782. doi:10.1038/nprot.2016.190.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-002D-0938-1 Version Permalink: http://hdl.handle.net/21.11116/0000-0001-218D-B
Genre: Journal Article

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Fricke, P., Author
Chevelkov, V., Author
Zinke, M., Author
Giller, K.1, Author              
Becker, S.1, Author              
Lange, A., Author
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1Department of NMR-Based Structural Biology, MPI for biophysical chemistry, Max Planck Society, ou_578567              

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 Abstract: Solid-state NMR (ssNMR) is a technique that allows the study of protein structure and dynamics at atomic detail. In contrast to X-ray crystallography and cryo-electron microscopy, proteins can be studied under physiological conditions-for example, in a lipid bilayer and at room temperature (0-35 degrees C). However, ssNMR requires considerable amounts (milligram quantities) of isotopically labeled samples. In recent years, H-1-detection of perdeuterated protein samples has been proposed as a method of alleviating the sensitivity issue. Such methods are, however, substantially more demanding to the spectroscopist, as compared with traditional C-13-detected approaches. As a guide, this protocol describes a procedure for the chemical shift assignment of the backbone atoms of proteins in the solid state by 1H-detected ssNMR. It requires a perdeuterated, uniformly 13C-and N-15-labeled protein sample with subsequent proton back-exchange to the labile sites. The sample needs to be spun at a minimum of 40 kHz in the NMR spectrometer. With a minimal set of five 3D NMR spectra, the protein backbone and some of the side-chain atoms can be completely assigned. These spectra correlate resonances within one amino acid residue and between neighboring residues; taken together, these correlations allow for complete chemical shift assignment via a ` backbone walk'. This results in a backbone chemical shift table, which is the basis for further analysis of the protein structure and/ or dynamics by ssNMR. Depending on the spectral quality and complexity of the protein, data acquisition and analysis are possible within 2 months.

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Language(s): eng - English
 Dates: 2017-03-092017-04
 Publication Status: Published in print
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 Rev. Method: Peer
 Identifiers: DOI: 10.1038/nprot.2016.190
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Title: Nature Protocols
Source Genre: Journal
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Pages: - Volume / Issue: 12 (4) Sequence Number: - Start / End Page: 764 - 782 Identifier: -