77Se enrichment of proteins expands the biological NMR toolbox

Stephanie Ramadan, Ming Dong, Renee P. Rubenstein, Wayne A. Wilkie, Brian J. Bahnson, Colin Thorpe, Sharon Rozovsky

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

Sulfur, a key contributor to biological reactivity, is not amendable to investigations by biological NMR spectroscopy. To utilize selenium as a surrogate, we have developed a generally applicable 77Se isotopic enrichment method for heterologous proteins expressed in Escherichia coli. We demonstrate 77Se NMR spectroscopy of multiple selenocysteine and selenomethionine residues in the sulfhydryl oxidase augmenter of liver regeneration (ALR). The resonances of the active-site residues were assigned by comparing the NMR spectra of ALR bound to oxidized and reduced flavin adenine dinucleotide. An additional resonance appears only in the presence of the reducing agent and disappears readily upon exposure to air and subsequent reoxidation of the flavin. Hence, 77Se NMR spectroscopy can be used to report the local electronic environment of reactive and structural sulfur sites, as well as changes taking place in those locations during catalysis.

Original languageEnglish
Pages (from-to)222-231
Number of pages10
JournalJournal of Molecular Biology
Volume425
Issue number2
DOIs
Publication statusPublished - 23 Jan 2013
Externally publishedYes

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Keywords

  • Se NMR
  • augmenter of liver regeneration
  • selenium NMR
  • selenocysteine
  • selenoproteins

ASJC Scopus subject areas

  • Molecular Biology

Cite this

Ramadan, S., Dong, M., Rubenstein, R. P., Wilkie, W. A., Bahnson, B. J., Thorpe, C., & Rozovsky, S. (2013). 77Se enrichment of proteins expands the biological NMR toolbox. Journal of Molecular Biology, 425(2), 222-231. https://doi.org/10.1016/j.jmb.2012.11.011