Specific 13C labeling of leucine, valine and isoleucine methyl groups for unambiguous detection of long-range restraints in protein solid-state NMR studies
Specific 13C labeling of leucine, valine and isoleucine methyl groups for unambiguous detection of long-range restraints in protein solid-state NMR studies
Publication date: Available online 6 January 2015 Source:Journal of Magnetic Resonance
Author(s): Hannes Klaus Fasshuber , Jean-Philippe Demers , Veniamin Chevelkov , Karin Giller , Stefan Becker , Adam Lange
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, ?-ketoisovalerate and ?-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. Graphical abstract
Scrambling free combinatorial labeling of alanine-β, isoleucine-δ1, leucine-pro S and valine-pro S methyl groups for the detection of long range NOEs
Scrambling free combinatorial labeling of alanine-β, isoleucine-δ1, leucine-pro S and valine-pro S methyl groups for the detection of long range NOEs
Abstract
Specific isotopic labeling of methyl groups in proteins has greatly extended the applicability of solution NMR spectroscopy. Simultaneous labeling of the methyl groups of several different amino acid types can offer a larger number of useful probes that can be used for structural characterisations of challenging proteins. Herein, we propose an improved AILV methyl-labeling protocol in which L...
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[NMR paper] Unambiguous Assignment of Short- and Long-Range Structural Restraints by Solid-State NMR Spectroscopy with Segmental Isotope Labeling.
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Abstract
A methyl-detected 'out-and-back' NMR experiment for obtaining simultaneous correlations of methyl resonances of valine and isoleucine/leucine residues with backbone carbonyl chemical shifts,...
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http://www.bionmr.com//www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--production.springer.de-OnlineResources-Logos-springerlink.gif Related Articles Specific labeling and assignment strategies of valine methyl groups for NMR studies of high molecular weight proteins.
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The specific protonation of valine and leucine methyl...
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Authors: Ayala I, Hamelin O, Amero C, Pessey O, Plevin MJ, Gans P, Boisbouvier J
An efficient synthetic route is proposed to produce 2-hydroxy-2-ethyl-3-oxobutanoate for the specific labelling of Ile methyl-?(2) groups in proteins. The (2)H,...
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J Am Chem Soc. 2011 Mar 24;
Authors: Linser R, Bardiaux B, Higman V, Fink U, Reif B
Magic-angle spinning (MAS) solid-state NMR becomes an increasingly important tool for the determination of structures of membrane...
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Structure Calculation from Unambiguous Long-Range Amide and Methyl 1H-1H Distance Restraints for a Microcrystalline Protein with MAS Solid-State NMR Spectroscopy
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http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jacsat/0/jacsat.ahead-of-print/ja110222h/aop/images/medium/ja-2010-10222h_0004.gif
Journal of the American Chemical Society
DOI: 10.1021/ja110222h
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http://feeds.feedburner.com/~r/acs/jacsat/~4/Dh0EBf8PwcY
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J Biomol Struct Dyn. 2003 Oct;21(2):235-46
Authors: Salopek-Sondi B, Vaughan MD, Skeels MC, Honek JF, Luck LA
The leucine-isoleucine-valine binding protein (LIV) found in the periplasmic space of E. coli has been used as a structural model for a number of neuronal receptors. This...
Specific 13C labeling of leucine, valine and isoleucine methyl groups for unambiguous detection of long-range restraints in protein solid-state NMR studies
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