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Engineering heme binding sites in monomeric rop.

Giovanna Di Nardo1, Almerinda Di Venere, Giampiero Mei

  • 1Department of Human and Animal Biology, University of Turin, Turin, Italy.

Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry
|January 20, 2009
PubMed
Summary
This summary is machine-generated.

Engineered repressor of primer (rop) mutants bind heme in distinct hydrophobic core layers. Layer 3 mutants exhibit tighter heme binding and increased protein stability compared to layer 1 mutants.

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Area of Science:

  • Biochemistry
  • Protein Engineering
  • Spectroscopy

Background:

  • The repressor of primer (rop) protein is crucial for plasmid replication.
  • Engineering hydrophobic cores of proteins can alter their functional properties.
  • Heme incorporation into proteins can modulate their stability and redox characteristics.

Purpose of the Study:

  • To engineer heme-binding sites within the hydrophobic core of a monomeric ColE1 repressor of primer (rop-S55).
  • To investigate the impact of heme ligand position on protein stability, binding affinity, and redox potential.
  • To compare heme binding in different layers of the protein's heptad repeat structure.

Main Methods:

  • Site-directed mutagenesis to introduce histidine residues for heme coordination.
  • Spectroscopic techniques (circular dichroism, fluorescence) to monitor protein unfolding.
  • Spectroelectrochemical titrations to determine heme reduction potentials.
  • Isothermal titration calorimetry or similar methods to quantify binding affinity (implied by K(D) values).

Main Results:

  • Two rop mutants, rop-L63M/F121H (layer 1) and rop-L56H/L113H (layer 3), were successfully engineered to bind heme.
  • Layer 3 mutant showed tighter heme binding (K(D) = 0.47 +/- 0.07 microM) compared to layer 1 mutant (K(D) = 1.1 +/- 0.2 microM).
  • Heme binding increased protein stability ([DGobsH2O]) by 1.4 kcal/mol (layer 1) and 1.8 kcal/mol (layer 3), with layer 3 mutant being more stable.
  • Reduction potentials differed significantly: -87.5 +/- 1.2 mV for layer 1 and -154 +/- 2 mV for layer 3.

Conclusions:

  • The location of the engineered heme-binding site within the hydrophobic core significantly influences heme binding affinity and protein stability.
  • A more buried heme-binding environment (layer 3) leads to enhanced protein stability and tighter heme association.
  • The redox properties of the bound heme are sensitive to the microenvironment within the protein core.