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PEDS Advance Access originally published online on October 24, 2007
Protein Engineering Design and Selection 2007 20(10):511-520; doi:10.1093/protein/gzm053
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Enhancing the thermal stability of mitochondrial cytochrome b5 by introducing a structural motif characteristic of the less stable microsomal isoform

Lijun Wang, Aaron B. Cowley and David R. Benson1

Department of Chemistry, University of Kansas, Lawrence, KS 66045, USA

1 To whom correspondence should be addressed. E-mail: drb{at}ku.edu

Outer mitochondrial membrane cytochrome b5 (OM b5) is the most thermostable cytochrome b5 isoform presently known. Herein, we show that OM b5 thermal stability is substantially enhanced by swapping an apparently invariant motif in its heme-independent folding core with the corresponding motif characteristic of its less stable evolutionary relative, microsomal cytochrome b5 (Mc b5). The motif swap involved replacing two residues, Arg15 with His and Glu20 with Ser, thereby introducing a Glu11-His15-Ser20 H-bonding triad on the protein surface along with a His15/Trp22 {pi}-stacking interaction. The ferric and ferrous forms of the OM b5 R15H/E20S double mutant have thermal denaturation midpoints (Tm values) of ~93°C and ~104°C, respectively. A 15°C increase in apoprotein Tm plays a key role in the holoprotein thermal stability enhancement, and is achieved by one of the most common natural mechanisms for stabilization of thermophilic versus mesophilic proteins: raising the unfolding free energy along the entire stability curve.

Keywords: apoproteins/cytochrome b5/m values/residual structure/thermal stability

Received March 13, 2007; revised September 12, 2007; accepted September 14, 2007.


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