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Publication Abstract from PubMed

Here we present the preparation, biophysical characterization, and nuclear magnetic resonance (NMR) spectroscopy study of yeast cytochrome c peroxidase (CcP) constructs with enhanced solubility. Using a high-yield Escherichia coli expression system, we routinely produced uniformly labeled [(2)H,(13)C,(15)N]CcP samples with high levels of deuterium incorporation (96-99%) and good yields (30-60 mg of pure protein from 1 L of bacterial culture). In addition to simplifying the purification procedure, introduction of a His tag at either protein terminus dramatically increases its solubility, allowing preparation of concentrated, stable CcP samples required for multidimensional NMR spectroscopy. Using a range of biophysical techniques and X-ray crystallography, we demonstrate that the engineered His tags neither perturb the structure of the enzyme nor alter the heme environment or its reactivity toward known ligands. The His-tagged CcP constructs remain catalytically active yet exhibit differences in the interaction with cytochrome c, the physiological binding partner, most likely because of steric occlusion of the high-affinity binding site by the C-terminal His tag. We show that protein perdeuteration greatly increases the quality of the double- and triple-resonance NMR spectra, allowing nearly complete backbone resonance assignments and subsequent study of the CcP by heteronuclear NMR spectroscopy.

Expression, purification, characterization, and solution nuclear magnetic resonance study of highly deuterated yeast cytochrome C peroxidase with enhanced solubility.,Volkov AN, Wohlkonig A, Soror SH, van Nuland NA Biochemistry. 2013 Apr 2;52(13):2165-75. doi: 10.1021/bi400220w. Epub 2013 Mar, 21. PMID:23517193^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.