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  Specific 13C labeling of leucine, valine and isoleucine methyl groups for unambiguous detection of long-range restraints in protein solid-state NMR studies.

Fasshuber, H. K., Demers, J. P., Chevelkov, V., Giller, K., Becker, S., & Lange, A. (2015). Specific 13C labeling of leucine, valine and isoleucine methyl groups for unambiguous detection of long-range restraints in protein solid-state NMR studies. Journal of Magnetic Resonance, 252, 10-19. doi:10.1016/j.jmr.2014.12.013.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0024-B3E6-0 Version Permalink: http://hdl.handle.net/11858/00-001M-0000-002A-0E38-F
Genre: Journal Article

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 Creators:
Fasshuber, H. K.1, Author              
Demers, J. P.1, Author              
Chevelkov, V.1, Author              
Giller, K.2, Author              
Becker, S.2, Author              
Lange, A.2, Author              
Affiliations:
1Research Group of Solid-State NMR, MPI for biophysical chemistry, Max Planck Society, ou_persistent35              
2Department of NMR-Based Structural Biology, MPI for biophysical chemistry, Max Planck Society, ou_578567              

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Free keywords: (13)C methyl labeling; Distance restraints; Dynamics; Nuclear Magnetic Resonance; Protein structure; Proton detection; Solid-state NMR; α-Ketobutyrate; α-Ketoisovalerate
 Abstract: Here we present an isotopic labeling strategy to easily obtain unambiguous long-range distance restraints in protein solid-state NMR studies. The method is based on the inclusion of two biosynthetic precursors in the bacterial growth medium, α-ketoisovalerate and α-ketobutyrate, leading to the production of leucine, valine and isoleucine residues that are exclusively 13C labeled on methyl groups. The resulting spectral simplification facilitates the collection of distance restraints, the verification of carbon chemical shift assignments and the measurement of methyl group dynamics. This approach is demonstrated on the type-three secretion system needle of Shigella flexneri, where 49 methyl-methyl and methyl-nitrogen distance restraints including 10 unambiguous long-range distance restraints could be collected. By combining this labeling scheme with ultra-fast MAS and proton detection, the assignment of methyl proton chemical shifts was achieved.

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Language(s): eng - English
 Dates: 2015-01-062015-03
 Publication Status: Published in print
 Pages: -
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 Rev. Method: Peer
 Identifiers: DOI: 10.1016/j.jmr.2014.12.013
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Title: Journal of Magnetic Resonance
Source Genre: Journal
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Pages: - Volume / Issue: 252 Sequence Number: - Start / End Page: 10 - 19 Identifier: -