Efficient identification of amino acid types for fast protein backbone assignments

H. D. Ou, H. C. Lai, Z. Serber, V. Dötsch

Research output: Contribution to journalArticle

28 Scopus citations

Abstract

We describe a procedure that allows for very efficient identification of amino acid types in proteins by selective 15N-labeling. The usefulness of selective incorporation of 15N-labeled amino acids into proteins for the backbone assignment has been recognized for several years. However, widespread use of this method has been hindered by the need to purify each selectively labeled sample and by the relatively high cost of labeling with 15N-labeled amino acids. Here we demonstrate that purification of the selectively 15N-labeled samples is not necessary and that background-free HSQC spectra containing only the peaks of the overexpressed heterologous protein can be obtained in crude lysates from as little as 100 ml cultures, thus saving time and money. This method can be used for fast and automated backbone assignment of proteins.

Original languageEnglish (US)
Pages (from-to)269-273
Number of pages5
JournalJournal of Biomolecular NMR
Volume21
Issue number3
DOIs
StatePublished - Dec 24 2001

Keywords

  • Automated assignment
  • Calmodulin
  • HSQC
  • SAM-domain
  • Selective labeling

ASJC Scopus subject areas

  • Biochemistry
  • Spectroscopy

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