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(77)Se enrichment of proteins expands the biological NMR toolbox.

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 (77)Se isotopic enrichment method for heterologous proteins expressed in Escherichia coli. We demonstrate (77)Se 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, (77)Se 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.

SUBMITTER: Schaefer SA

PROVIDER: S-EPMC3540199 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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(77)Se enrichment of proteins expands the biological NMR toolbox.

Schaefer Stephanie A SA Dong Ming M Rubenstein Renee P RP Wilkie Wayne A WA Bahnson Brian J BJ Thorpe Colin C Rozovsky Sharon S

Journal of molecular biology 20121115 2

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 (77)Se isotopic enrichment method for heterologous proteins expressed in Escherichia coli. We demonstrate (77)Se 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 assi ...[more]

PMID: 23159557

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