Helix mutations stabilize a late productive intermediate on the folding pathway of ubiquitin

Biochemistry. 2008 Aug 5;47(31):8225-36. doi: 10.1021/bi800722d. Epub 2008 Jul 11.


We have investigated the relative placement of rate-limiting energy barriers and the role of productive or obstructive intermediates on the folding pathway of yeast wild-type ubiquitin ( wt-Ub) containing the F45W mutation. To manipulate the folding barriers, we have designed a family of mutants in which stabilizing substitutions have been introduced incrementally on the solvent-exposed surface of the main alpha-helix (residues 23-34), which has a low intrinsic helical propensity in the native sequence. Although the U --> I and I --> N transitions are not clearly delineated in the kinetics of wt-Ub, we show that an intermediate becomes highly populated and more clearly resolved as the predicted stability of the helix increases. The observed acceleration in the rate of folding correlates with helix stability and is consistent with the I-state representing a productive rather than misfolded state. A Leffler analysis of the effects on kinetics of changes in stability within the family of helix mutants results in a biphasic correlation in both the refolding and unfolding rates that suggest a shift from a nucleation-condensation mechanism (weakly stabilized helix) toward a diffusion-collision model (highly stabilized helix). Through the introduction of helix-stabilizing mutations, we are able to engineer a well-resolved I-state on the folding pathway of ubiquitin which is likely to be structurally distinct from that which is only weakly populated on the folding pathway of wild-type ubiquitin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Circular Dichroism
  • Kinetics
  • Magnetic Resonance Spectroscopy
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutation*
  • Protein Denaturation
  • Protein Folding
  • Protein Structure, Secondary
  • Sequence Homology, Amino Acid
  • Temperature
  • Thermodynamics
  • Ubiquitin / chemistry*
  • Ubiquitin / genetics*


  • Ubiquitin