Protein vivisection reveals elusive intermediates in folding

J Mol Biol. 2010 Apr 2;397(3):777-88. doi: 10.1016/j.jmb.2010.01.056. Epub 2010 Feb 6.

Abstract

Although most folding intermediates escape detection, their characterization is crucial to the elucidation of folding mechanisms. Here, we outline a powerful strategy to populate partially unfolded intermediates: A buried aliphatic residue is substituted with a charged residue (e.g., Leu-->Glu(-)) to destabilize and unfold a specific region of the protein. We applied this strategy to ubiquitin, reversibly trapping a folding intermediate in which the beta5-strand is unfolded. The intermediate refolds to a native-like structure upon charge neutralization under mildly acidic conditions. Characterization of the trapped intermediate using NMR and hydrogen exchange methods identifies a second folding intermediate and reveals the order and free energies of the two major folding events on the native side of the rate-limiting step. This general strategy may be combined with other methods and have broad applications in the study of protein folding and other reactions that require trapping of high-energy states.

Publication types

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

MeSH terms

  • Deuterium Exchange Measurement
  • Models, Chemical
  • Models, Molecular*
  • Nuclear Magnetic Resonance, Biomolecular
  • Protein Folding*
  • Scattering, Radiation
  • Thermodynamics
  • Ubiquitin / chemistry*
  • Ubiquitin / genetics
  • Ubiquitin / metabolism

Substances

  • Ubiquitin