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

    Research output: Contribution to journalArticle

    16 Citations (Scopus)

    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.

    Original languageEnglish
    Pages (from-to)167-174
    Number of pages8
    JournalJournal of Structural and Functional Genomics
    Volume12
    Issue number3
    DOIs
    Publication statusPublished - Sep 1 2011

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    All Science Journal Classification (ASJC) codes

    • Structural Biology
    • Biochemistry
    • Genetics

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