PDBsum entry 4jb4

Oxidoreductase

PDB id: 4jb4

Contents

Protein chains

291 a.a.

Ligands

HEM ×2

Metals

__F ×2

Waters ×36

References listed in PDB file

Key reference

• Title: Expression, Purification, Characterization, And solution nuclear magnetic resonance study of highly deuterated yeast cytochrome c peroxidase with enhanced solubility.
• Authors: A.N.Volkov, A.Wohlkonig, S.H.Soror, N.A.Van nuland.
• Ref.: Biochemistry, 2013, 52, 2165-2175. [DOI no: 10.1021/bi400220w]
• PubMed id: 23517193

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.