A simplified recipe for assigning amide NMR signals using combinatorial 14N amino acid inverse-labeling
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.
KeywordsCombinatorial inverse-labeling Aβ(1–40) peptide NMR sample preparation Isotope labeling
Heteronuclear single quantum coherence spectroscopy
Band-selective optimized flip-angle short-transient heteronuclear multiple quantum coherence spectroscopy
Band-selective excitation short-transient
Transverse relaxation optimized correlation spectroscopy
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.
- 2.Pervushin K, Riek R, Wider G, Wuthrich K (1997) Attenuated T2 relaxation by mutual cancellation of dipole–dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc Natl Acad Sci USA 94:12366–12371PubMedCrossRefGoogle Scholar
- 3.Cavanagh J, Fairbrother WJ, Palmer AG III, Skelton NJ (2007) Protein NMR spectroscopy: principles and practice, 2nd edn. Academic Press, San Diego, CAGoogle Scholar
- 4.Ikura M, Kay LE, Bax A (1990) A novel approach for sequential assignment of 1H, 13C and 15N spectra of proteins: heteronuclear triple-resonance three-dimensional NMR spectroscopy. Appl Calmodulin Biochem 29:4659–4667Google Scholar
- 7.Barna JCJ, Laue ED, Mayger MR, Skilling J, Worrall SJP (1987) Exponential sampling an alternative method for sampling in two-dimensional NMR experiments. J Magn Reson 73:69–77Google Scholar
- 29.Yokochi M, Sekiguchi S, Inagaki F (2010) Olivia. Hokkaido University, SapporoGoogle Scholar