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

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.

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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

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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.

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Correspondence to Hidekazu Hiroaki.

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Hiroaki, H., Umetsu, Y., Nabeshima, Y. et al. A simplified recipe for assigning amide NMR signals using combinatorial 14N amino acid inverse-labeling. J Struct Funct Genomics 12, 167 (2011). https://doi.org/10.1007/s10969-011-9116-0

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Keywords

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