Rational Design of Domain-Swapping-Based c-Type Cytochrome Heterodimers by Using Chimeric Proteins

Chembiochem. 2017 Sep 5;18(17):1712-1715. doi: 10.1002/cbic.201700219. Epub 2017 Jul 21.

Abstract

The design of protein oligomers with multiple active sites has been gaining interest, owing to their potential use for biomaterials, which has encouraged researchers to develop a new design method. Three-dimensional domain swapping is the unique phenomenon in which protein molecules exchange the same structural region between each other. Herein, to construct oligomeric heme proteins with different active sites by utilizing domain swapping, two c-type cytochrome-based chimeric proteins have been constructed and the domains swapped. According to X-ray crystallographic analysis, the two chimeric proteins formed a domain-swapped dimer with two His/Met coordinated hemes. By mutating the heme coordination structure of one of the two chimeric proteins, a domainswapped heterodimer with His/Met and His/H2 O coordinated hemes was formed. Binding of an oxygen molecule to the His/H2 O site of the heterodimer was confirmed by resonance Raman spectroscopy, in which the Fe-O2 stretching band was observed at 580 cm-1 for the reduced/oxygenated heterodimer (at 554 cm-1 under an 18 O2 atmosphere). These results show that domain swapping is a useful method to design multiheme proteins.

Keywords: cytochromes; domain swapping; heme proteins; oligomers; protein design.

MeSH terms

  • Aquifoliaceae / enzymology
  • Circular Dichroism
  • Crystallography, X-Ray
  • Cytochrome c Group / chemistry
  • Cytochrome c Group / genetics
  • Cytochrome c Group / metabolism*
  • Dimerization
  • Heme / chemistry
  • Heme / metabolism
  • Oxygen / chemistry
  • Protein Engineering
  • Protein Structure, Tertiary
  • Pseudomonas aeruginosa / enzymology
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / isolation & purification
  • Spectrum Analysis, Raman

Substances

  • Cytochrome c Group
  • Recombinant Fusion Proteins
  • Heme
  • Oxygen