|Title||Expression, purification, characterization, and solution nuclear magnetic resonance study of highly deuterated yeast cytochrome C peroxidase with enhanced solubility.|
|Publication Type||Journal Article|
|Year of Publication||2013|
|Authors||Volkov, A., A. Wohlkonig, S. H. Soror, and N. A. J. van Nuland|
|Date Published||2013 Apr 2|
|Keywords||Amino Acid Sequence, Circular Dichroism, Cloning, Molecular, Crystallography, X-Ray, Cytochrome-c Peroxidase, Cytochromes c, Escherichia coli, Gene Expression, Kinetics, Models, Molecular, Molecular Sequence Data, Nuclear Magnetic Resonance, Biomolecular, Protein Binding, Saccharomyces cerevisiae, Solubility, Spectrometry, Mass, Electrospray Ionization|
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.