A conserved folding nucleus sculpts the free energy landscape of bacterial and archaeal orthologs from a divergent TIM barrel family

Proc Natl Acad Sci U S A. 2021 Apr 27;118(17):e2019571118. doi: 10.1073/pnas.2019571118.


The amino acid sequences of proteins have evolved over billions of years, preserving their structures and functions while responding to evolutionary forces. Are there conserved sequence and structural elements that preserve the protein folding mechanisms? The functionally diverse and ancient (βα)1-8 TIM barrel motif may answer this question. We mapped the complex six-state folding free energy surface of a ∼3.6 billion y old, bacterial indole-3-glycerol phosphate synthase (IGPS) TIM barrel enzyme by equilibrium and kinetic hydrogen-deuterium exchange mass spectrometry (HDX-MS). HDX-MS on the intact protein reported exchange in the native basin and the presence of two thermodynamically distinct on- and off-pathway intermediates in slow but dynamic equilibrium with each other. Proteolysis revealed protection in a small (α1β2) and a large cluster (β5α5β6α6β7) and that these clusters form cores of stability in Ia and Ibp The strongest protection in both states resides in β4α4 with the highest density of branched aliphatic side chain contacts in the folded structure. Similar correlations were observed previously for an evolutionarily distinct archaeal IGPS, emphasizing a key role for hydrophobicity in stabilizing common high-energy folding intermediates. A bioinformatics analysis of IGPS sequences from the three superkingdoms revealed an exceedingly high hydrophobicity and surprising α-helix propensity for β4, preceded by a highly conserved βα-hairpin clamp that links β3 and β4. The conservation of the folding mechanisms for archaeal and bacterial IGPS proteins reflects the conservation of key elements of sequence and structure that first appeared in the last universal common ancestor of these ancient proteins.

Keywords: TIM barrel orthologs; hydrogen deuterium exchange; mass spectrometry; protein evolution; protein folding.

Publication types

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

MeSH terms

  • Amino Acid Sequence / genetics
  • Amino Acids / genetics
  • Bacterial Proteins / chemistry
  • Hydrogen Bonding
  • Indole-3-Glycerol-Phosphate Synthase / metabolism*
  • Indole-3-Glycerol-Phosphate Synthase / physiology
  • Kinetics
  • Models, Molecular
  • Protein Conformation
  • Protein Domains / genetics
  • Protein Domains / physiology*
  • Protein Folding
  • Protein Structure, Secondary / genetics*
  • Sequence Homology, Amino Acid
  • Thermodynamics


  • Amino Acids
  • Bacterial Proteins
  • Indole-3-Glycerol-Phosphate Synthase