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Converting a maltose receptor into a nascent binuclear copper oxygenase by computational design
David E Benson1, Alice E Haddy, Homme W Hellinga
1Department of Chemistry, 221 Petty Building, University of North Carolina-Greensboro, Greensboro, North Carolina 27402, USA.
Biochemistry
|February 28, 2002
Summary
Computational protein design created a novel oxygen-binding site in maltose-binding protein (MBP). One mutant protein successfully binds copper and cobalt, mimicking aspects of oxy-hemocyanin with hydrogen peroxide.
Area of Science:
- Biochemistry
- Structural Biology
- Protein Engineering
Background:
- Maltose-binding protein (MBP) from Escherichia coli typically binds maltose.
- Computational methods are advancing protein design for novel functions.
Purpose of the Study:
- To engineer a binuclear copper-binding site into MBP for oxygen binding.
- To investigate the functional properties of engineered metalloprotein complexes.
Main Methods:
- Computational protein design to identify mutations.
- Oligonucleotide-directed mutagenesis to construct mutant proteins.
- Protein expression, purification, and characterization of metal-binding and reactivity.
Main Results:
- Five candidate designs with 9-10 mutations were created.
- One mutant, MBP.Hc.E, formed functional Cu(II)2 and Co(II)2 complexes.
- The Co(II)2 complex showed spectroscopic similarity to oxy-hemocyanin models.
- A further mutation (I329F) enhanced mimicry of oxy-hemocyanin's spectroscopic signature.
Conclusions:
- Engineered binuclear metal centers can be introduced into MBP.
- The engineered protein can bind oxygen and mimic hemocyanin's active site.
- Protein conformational flexibility influences ligand binding in engineered metalloproteins.