77Se Enrichment of Proteins Expands the Biological NMR Toolbox

被引：24

作者：

Schaefer, Stephanie A. ^{[1
]}

Dong, Ming ^{[1
]}

Rubenstein, Renee P. ^{[1
]}

Wilkie, Wayne A. ^{[1
]}

Bahnson, Brian J. ^{[1
]}

Thorpe, Colin ^{[1
]}

Rozovsky, Sharon ^{[1
]}

机构：

[1] Univ Delaware, Dept Chem & Biochem, Brown Lab 136, Newark, DE 19716 USA

来源：

JOURNAL OF MOLECULAR BIOLOGY | 2013年 / 425卷 / 02期

基金：

美国国家科学基金会; 美国国家卫生研究院;

关键词：

X-RAY-DIFFRACTION; LIVER-REGENERATION; ESCHERICHIA-COLI; SULFHYDRYL OXIDASE; SELENOCYSTEINE; SELENIUM; DISELENIDE; AUGMENTOR; BIOSYNTHESIS; EXPRESSION;

D O I：

10.1016/j.jmb.2012.11.011

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

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 Se-77 isotopic enrichment method for heterologous proteins expressed in Escherichia coli. We demonstrate Se-77 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, Se-77 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. (C) 2012 Elsevier Ltd. All rights reserved.

引用

页码：222 / 231

页数：10

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