Divergent evolution of a bifunctional de novo protein

Betsy A. Smith, Ann E. Mularz, Michael H. Hecht

Research output: Contribution to journalArticlepeer-review

20 Scopus citations


Primordial proteins, the evolutionary ancestors of modern sequences, are presumed to have been minimally active and nonspecific. Following eons of selective pressure, these early progenitors evolved into highly active and specific proteins. While evolutionary trajectories from poorly active and multifunctional generalists toward highly active specialists likely occurred many times in evolutionary history, such pathways are difficult to reconstruct in natural systems, where primordial sequences are lost to time. To test the hypothesis that selection for enhanced activity leads to a loss of promiscuity, we evolved a de novo designed bifunctional protein. The parental protein, denoted Syn-IF, was chosen from a library of binary patterned 4-helix bundles. Syn-IF was shown previously to rescue two different auxotrophic strains of E. coli: ΔilvA and Δfes. These two strains contain deletions for proteins with very different biochemical functions; IlvA is involved in isoleucine biosynthesis, while Fes is involved in iron assimilation. In two separate experiments, Syn-IF, was evolved for faster rescue of either ΔilvA or Δfes. Following multiple rounds of mutagenesis, two new proteins were selected, each capable of rescuing the selected function significantly faster than the parental protein. In each case, the evolved protein also lost the ability to rescue the unselected function. In both evolutionary trajectories, the original bifunctional generalist was evolved into a monofunctional specialist with enhanced activity.

Original languageEnglish (US)
Pages (from-to)246-252
Number of pages7
JournalProtein Science
Issue number2
StatePublished - Feb 1 2015

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Biochemistry


  • bifunctional protein
  • binary code
  • directed evolution
  • four helix bundle
  • functional promiscuity
  • polar/nonpolar patterning
  • protein design


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