title: Expression, purification, characterization, and solution nuclear magnetic resonance study of highly deuterated yeast cytochrome C peroxidase with enhanced solubility.
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.
authors.affiliation: Jean Jeener NMR Centre, Structural Biology Brussels, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium. ovolkov@vub.ac.be
authors.forename: Alexander N
authors.initials: AN
authors.lastname: Volkov
authors.forename: Alexandre
authors.initials: A
authors.lastname: Wohlkonig
authors.forename: Sameh H
authors.initials: SH
authors.lastname: Soror
authors.forename: Nico A J
authors.initials: NA
authors.lastname: van Nuland
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D003578:Cytochrome-c Peroxidase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D045304:Cytochromes c; D003578:Cytochrome-c Peroxidase