De novo design of a fluorescence-activating β-barrel

Nature. 2018 Sep;561(7724):485-491. doi: 10.1038/s41586-018-0509-0. Epub 2018 Sep 12.


The regular arrangements of β-strands around a central axis in β-barrels and of α-helices in coiled coils contrast with the irregular tertiary structures of most globular proteins, and have fascinated structural biologists since they were first discovered. Simple parametric models have been used to design a wide range of α-helical coiled-coil structures, but to date there has been no success with β-barrels. Here we show that accurate de novo design of β-barrels requires considerable symmetry-breaking to achieve continuous hydrogen-bond connectivity and eliminate backbone strain. We then build ensembles of β-barrel backbone models with cavity shapes that match the fluorogenic compound DFHBI, and use a hierarchical grid-based search method to simultaneously optimize the rigid-body placement of DFHBI in these cavities and the identities of the surrounding amino acids to achieve high shape and chemical complementarity. The designs have high structural accuracy and bind and fluorescently activate DFHBI in vitro and in Escherichia coli, yeast and mammalian cells. This de novo design of small-molecule binding activity, using backbones custom-built to bind the ligand, should enable the design of increasingly sophisticated ligand-binding proteins, sensors and catalysts that are not limited by the backbone geometries available in known protein structures.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Benzyl Compounds / analysis
  • Benzyl Compounds / chemistry*
  • COS Cells
  • Chlorocebus aethiops
  • Escherichia coli
  • Fluorescence*
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Hydrogen Bonding
  • Imidazolines / analysis
  • Imidazolines / chemistry*
  • Ligands
  • Protein Binding
  • Protein Domains
  • Protein Folding
  • Protein Stability
  • Protein Structure, Secondary
  • Proteins / chemistry*
  • Reproducibility of Results
  • Yeasts


  • 3,5-difluoro-4-hydroxybenzylidene imidazolinone
  • Benzyl Compounds
  • Imidazolines
  • Ligands
  • Proteins
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins