A simplified recipe for assigning amide NMR signals using combinatorial 14N amino acid inverse-labeling

  • Hidekazu Hiroaki
  • Yoshitaka Umetsu
  • Yo-ichi Nabeshima
  • Minako Hoshi
  • Daisuke Kohda
Article

Abstract

Assignment of backbone amide proton resonances is one of the most time-consuming stages of any protein NMR study when the protein samples behave non-ideally. A robust and convenient NMR procedure for analyzing spectra of marginal-to-low quality is helpful for high-throughput structure determination. The 14N selective- and inverse-labeling method is a candidate solution. Here, we present a simplified protocol for assigning protein backbone amide NMR signals. When 14N inversely labeled residues are present in a protein, their backbone NH cross peaks vanish from the protein’s 1H–15N HSQC spectrum, and thus, their chemical shifts can be readily identified by a process of elimination. Some metabolically related amino acids, for example, Ile, Leu, and Val, cannot be individually incorporated but can be inversely labeled together. We optimized and simplified the protocol and M9-based medium formula for the 14N selective- and inverse-labeling method without any additives. Our approach should be cost-effective, because the method could be additively applied stepwise, even when the proteins of interest were found to be non-ideal.

Keywords

Combinatorial inverse-labeling Aβ(1–40) peptide NMR sample preparation Isotope labeling 

Abbreviations

HSQC

Heteronuclear single quantum coherence spectroscopy

IPTG

Isopropyl-β-d-galactoside

IL1β

Interleukin-1β

SOFAST-HMQC

Band-selective optimized flip-angle short-transient heteronuclear multiple quantum coherence spectroscopy

BEST

Band-selective excitation short-transient

TROSY

Transverse relaxation optimized correlation spectroscopy

Notes

Acknowledgments

We thank the people who helped to confirm the versatility of the method by applying it to each of the proteins: Dr. M. Shimizu (gcm-DBD), Mr. M. Itoh (LOV2phot), Dr. H. Tochio and Dr. H. Ohnishi (TIR-MyD88). We also thank Dr. D. Hamada for providing an Aβ expression system.

Supplementary material

10969_2011_9116_MOESM1_ESM.pdf (25 kb)
Supplementary material 1 (PDF 24 kb)
10969_2011_9116_MOESM2_ESM.pdf (195 kb)
Supplementary material 2 (PDF 194 kb)

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Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Hidekazu Hiroaki
    • 1
    • 2
    • 3
  • Yoshitaka Umetsu
    • 1
    • 2
  • Yo-ichi Nabeshima
    • 4
  • Minako Hoshi
    • 4
  • Daisuke Kohda
    • 3
    • 5
  1. 1.Division of Structural BiologyKobe University Graduate School of MedicineChuo, KobeJapan
  2. 2.The Structural Biology Resreach Center and Division of Biological Science, Graduate School of ScienceNagoya UniversityFuro-cho, Chikusa-ku, NagoyaJapan
  3. 3.Department of Structural BiologyBiomolecular Engineering Research InstituteSuita, OsakaJapan
  4. 4.Department of Pathology and Tumor BiologyKyoto University Graduate School of MedicineKyotoJapan
  5. 5.Medical Institute for BioregulationKyushu UniversityHakata, FukuokaJapan

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