Abstract
Heteronuclear metalloenzymes catalyze some of the most fundamentally interesting and practically useful reactions in nature. However, the presence of two or more metal ions in close proximity in these enzymes makes them more difficult to prepare and study than homonuclear metalloenzymes. To meet these challenges, heteronuclear metal centers have been designed into small and stable proteins with rigid scaffolds to understand how these heteronuclear centers are constructed and the mechanism of their function. This chapter describes methods for designing heterobinuclear metal centers in a protein scaffold by giving specific examples of a few heme–nonheme bimetallic centers engineered in myoglobin and cytochrome c peroxidase. We provide step-by-step procedures on how to choose the protein scaffold, design a heterobinuclear metal center in the protein scaffold computationally, incorporate metal ions into the protein, and characterize the resulting metalloproteins, both structurally and functionally. Finally, we discuss how an initial design can be further improved by rationally tuning its secondary coordination sphere, electron/proton transfer rates, and the substrate affinity.
Original language | English (US) |
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Title of host publication | Methods in Enzymology |
Publisher | Academic Press Inc. |
Pages | 501-537 |
Number of pages | 37 |
DOIs | |
State | Published - 2016 |
Externally published | Yes |
Publication series
Name | Methods in Enzymology |
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Volume | 580 |
ISSN (Print) | 0076-6879 |
ISSN (Electronic) | 1557-7988 |
Bibliographical note
Funding Information:We wish to thank all the former and current Lu group members who have contributed to the development of the protocols and obtaining results published in papers cited in this chapter. The Lu group research described in papers cited in this chapter has been supported by the US National Institute of Health (R01GM06211) and National Science Foundation (CHE 14-13328).
Publisher Copyright:
© 2016 Elsevier Inc.
Keywords
- Biomimetic models
- Biosynthetic models
- Heme–copper oxidase
- Manganese peroxidase
- Metalloprotein design
- Nitric oxide reductase
- Oxygen activation
- Protein design
- Protein engineering
- Secondary sphere interactions