title: Specific 13C labeling of leucine, valine and isoleucine methyl groups for unambiguous detection of long-range restraints in protein solid-state NMR studies.
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 (13)C 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.
authors.affiliation: Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany; Department of Molecular Biophysics, Leibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Rössle-Str. 10, 13125 Berlin, Germany.
authors.forename: Hannes Klaus
authors.initials: HK
authors.lastname: Fasshuber
authors.affiliation: Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany; Department of Molecular Biophysics, Leibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Rössle-Str. 10, 13125 Berlin, Germany.
authors.forename: Jean-Philippe
authors.initials: JP
authors.lastname: Demers
authors.affiliation: Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany; Department of Molecular Biophysics, Leibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Rössle-Str. 10, 13125 Berlin, Germany.
authors.forename: Veniamin
authors.initials: V
authors.lastname: Chevelkov
authors.affiliation: Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
authors.forename: Karin
authors.initials: K
authors.lastname: Giller
authors.affiliation: Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Becker
authors.affiliation: Department of NMR-based Structural Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany; Department of Molecular Biophysics, Leibniz-Institut für Molekulare Pharmakologie (FMP), Robert-Rössle-Str. 10, 13125 Berlin, Germany; Institut für Biologie, Humboldt-Universität zu Berlin, Invalidenstr. 110, 10115 Berlin, Germany. Electronic address: alange@fmp-berlin.de.
authors.forename: Adam
authors.initials: A
authors.lastname: Lange
mesh_terms: D002247:Carbon Isotopes / Q000737:chemistry; D007532:Isoleucine / Q000032:analysis / Q000737:chemistry*; D007553:Isotope Labeling / Q000379:methods; D007930:Leucine / Q000032:analysis / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008745:Methylation; D010449:Peptide Mapping / Q000379:methods*; D011506:Proteins / Q000032:analysis / Q000737:chemistry*; D011522:Protons; D019275:Radiopharmaceuticals / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014633:Valine / Q000032:analysis / Q000737:chemistry*
chemical_list: D002247:Carbon Isotopes; D011506:Proteins; D011522:Protons; D019275:Radiopharmaceuticals; D007532:Isoleucine; D007930:Leucine; D014633:Valine
keywords: (13)C methyl labeling; Distance restraints; Dynamics; Nuclear Magnetic Resonance; Protein structure; Proton detection; Solid-state NMR; α-Ketobutyrate; α-Ketoisovalerate