4JB4

Expression, Purification, Characterization, and Solution NMR Study of Highly Deuterated Yeast Cytochrome c Peroxidase with Enhanced Solubility

Summary for 4JB4

• Entry DOI: 10.2210/pdb4jb4/pdb
• Descriptor: Cytochrome c peroxidase, mitochondrial, PROTOPORPHYRIN IX CONTAINING FE, FLUORIDE ION, ... (4 entities in total)
• Functional Keywords: cytochrome c peroxidase, oxidoreductase
• Biological source: Saccharomyces cerevisiae (Baker's yeast)
• Cellular location: Mitochondrion matrix: P00431
• Total number of polymer chains: 2
• Total formula weight: 70195.51

Authors

Wohlkonig, A.C. (deposition date: 2013-02-19, release date: 2013-04-10, Last modification date: 2023-09-20)

Primary citation

Volkov, A.N.,Wohlkonig, A.,Soror, S.H.,van Nuland, N.A.
Expression, purification, characterization, and solution nuclear magnetic resonance study of highly deuterated yeast cytochrome C peroxidase with enhanced solubility.
Biochemistry, 52:2165-2175, 2013

PubMed 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.

PubMed: 23517193
DOI: 10.1021/bi400220w

Experimental method

X-RAY DIFFRACTION (2.39 Å)