Design of a Fe4S4 cluster into the core of a de novo four-helix bundle

Joshua A. Mancini, Douglas H. Pike, Alexei M. Tyryshkin, Liti Haramaty, Michael S. Wang, Saroj Poudel, Michael Hecht, Vikas Nanda

Research output: Contribution to journalArticlepeer-review

6 Scopus citations


We explore the capacity of the de novo protein, S824, to incorporate a multinuclear iron–sulfur cluster within the core of a single-chain four-helix bundle. This topology has a high intrinsic designability because sequences are constrained largely by the pattern of hydrophobic and hydrophilic amino acids, thereby allowing for the extensive substitution of individual side chains. Libraries of novel proteins based on these constraints have surprising functional potential and have been shown to complement the deletion of essential genes in E. coli. Our structure-based design of four first-shell cysteine ligands, one per helix, in S824 resulted in successful incorporation of a cubane Fe4S4 cluster into the protein core. A number of challenges were encountered during the design and characterization process, including nonspecific metal-induced aggregation and the presence of competing metal-cluster stoichiometries. The introduction of buried iron–sulfur clusters into the helical bundle is an initial step toward converting libraries of designed structures into functional de novo proteins with catalytic or electron-transfer functionalities.

Original languageEnglish (US)
Pages (from-to)574-585
Number of pages12
JournalBiotechnology and Applied Biochemistry
Issue number4
StatePublished - Jul 1 2020

All Science Journal Classification (ASJC) codes

  • Applied Microbiology and Biotechnology
  • Drug Discovery
  • Bioengineering
  • Molecular Medicine
  • Process Chemistry and Technology
  • Biotechnology
  • Biomedical Engineering


  • combinatorial library
  • de novo protein
  • electron transfer
  • iron–sulfur cluster
  • oxidoreductase
  • protein design


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