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Default 77Se Enrichment of Proteins Expands the Biological NMR Toolbox

77Se Enrichment of Proteins Expands the Biological NMR Toolbox

23 January 2013
Publication year: 2013
Source:Journal of Molecular Biology, Volume 425, Issue 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 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.
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Highlights

? The NMR-active isotope 77Se is introduced into proteins by heterologous expression in E. coli. ? The method is cost effective and requires no synthesis or modifications to existing expression systems. ? The method permits control over the ratio of sulfur-to-selenium substitution in proteins allowing the manipulation of protein populations for medical and biochemical research. ? It is possible to resolve multiple selenomethionine and selenocysteine residues in the 32-kDa dimer of ALR by conventional NMR spectroscopy. ? Using the protein ALR, we further demonstrate the feasibility of routine biological Se NMR spectroscopy as a probe of structure and function.





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