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De Novo Design of Metalloproteins and Metalloenzymes in a Three-Helix Bundle

Part of the Methods in Molecular Biology book series (MIMB,volume 1414)

Abstract

For more than two decades, de novo protein design has proven to be an effective methodology for modeling native proteins. De novo design involves the construction of metal-binding sites within simple and/or unrelated α-helical peptide structures. The preparation of α3D, a single polypeptide that folds into a native-like three-helix bundle structure, has significantly expanded available de novo designed scaffolds. Devoid of a metal-binding site (MBS), we incorporated a 3Cys and 3His motif in α3D to construct a heavy metal and a transition metal center, respectively. These efforts produced excellent functional models for native metalloproteins/metalloregulatory proteins and metalloenzymes. Morever, these α3D derivatives serve as a foundation for constructing redox active sites with either the same (e.g., 4Cys) or mixed (e.g., 2HisCys) ligands, a feat that could be achieved in this preassembled framework. Here, we describe the process of constructing MBSs in α3D and our expression techniques.

Key words

  • De novo protein design
  • Three-helix bundle
  • Metal-binding site
  • Metalloprotein
  • Metalloregulatory protein
  • Metalloenzyme
  • Protein expression

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Acknowledgments

J.S.P. and V.L.P. would like to thank the National Institutes of Health (NIH) for financial support for this research (ES012236). J.S.P. thanks the Rackham Graduate School at the University of Michigan for a research fellowship.

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Correspondence to Vincent L. Pecoraro .

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Plegaria, J.S., Pecoraro, V.L. (2016). De Novo Design of Metalloproteins and Metalloenzymes in a Three-Helix Bundle. In: Stoddard, B. (eds) Computational Design of Ligand Binding Proteins. Methods in Molecular Biology, vol 1414. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3569-7_11

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  • DOI: https://doi.org/10.1007/978-1-4939-3569-7_11

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