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

15 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

Fingerprint

Liver Regeneration
Magnetic Resonance Spectroscopy
Sulfur
Selenocysteine
Selenomethionine
Proteins
Flavin-Adenine Dinucleotide
Reducing Agents
Selenium
Catalysis
Catalytic Domain
Air
Escherichia coli
sulfhydryl oxidase
4,6-dinitro-o-cresol

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

77Se enrichment of proteins expands the biological NMR toolbox. / Ramadan, Stephanie; Dong, Ming; Rubenstein, Renee P.; Wilkie, Wayne A.; Bahnson, Brian J.; Thorpe, Colin; Rozovsky, Sharon.

In: Journal of Molecular Biology, Vol. 425, No. 2, 23.01.2013, p. 222-231.

Research output: Contribution to journalArticle

Ramadan, S, Dong, M, Rubenstein, RP, Wilkie, WA, Bahnson, BJ, Thorpe, C & Rozovsky, S 2013, '77Se enrichment of proteins expands the biological NMR toolbox', Journal of Molecular Biology, vol. 425, no. 2, pp. 222-231. https://doi.org/10.1016/j.jmb.2012.11.011
Ramadan S, Dong M, Rubenstein RP, Wilkie WA, Bahnson BJ, Thorpe C et al. 77Se enrichment of proteins expands the biological NMR toolbox. Journal of Molecular Biology. 2013 Jan 23;425(2):222-231. https://doi.org/10.1016/j.jmb.2012.11.011
Ramadan, Stephanie ; Dong, Ming ; Rubenstein, Renee P. ; Wilkie, Wayne A. ; Bahnson, Brian J. ; Thorpe, Colin ; Rozovsky, Sharon. / 77Se enrichment of proteins expands the biological NMR toolbox. In: Journal of Molecular Biology. 2013 ; Vol. 425, No. 2. pp. 222-231.
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