Expression, purification, characterization, and solution nuclear magnetic resonance study of highly deuterated yeast cytochrome C peroxidase with enhanced solubility.

TitleExpression, purification, characterization, and solution nuclear magnetic resonance study of highly deuterated yeast cytochrome C peroxidase with enhanced solubility.
Publication TypeJournal Article
Year of Publication2013
AuthorsVolkov, A., A. Wohlkonig, S. H. Soror, and N. A. J. van Nuland
JournalBiochemistry
Volume52
Issue13
Pagination2165-75
Date Published2013 Apr 2
ISSN1520-4995
KeywordsAmino 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
Abstract

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.

DOI10.1021/bi400220w
Alternate JournalBiochemistry
PubMed ID23517193