Thermodynamic characterization of the folding equilibrium of the human Nedd4-WW4 domain: at the frontiers of cooperative folding

Biochemistry. 2009 Sep 15;48(36):8712-20. doi: 10.1021/bi9007758.

Abstract

WW domains are the smallest naturally independent beta-sheet protein structures available to date and constitute attractive model systems for investigating the determinants of beta-sheet folding and stability. Nonetheless, their small size and low cooperativity pose a difficult challenge for a quantitative analysis of the folding equilibrium. We describe here a comprehensive thermodynamic characterization of the conformational equilibrium of the fourth WW domain from the human ubiquitin ligase Nedd4 (hNedd4-WW4) using a combination of calorimetric and spectroscopic techniques with several denaturing agents (temperature, pH, and chemical denaturants). Our results reveal that even though the experimental data can be described in terms of a two-state equilibrium, spectral data together with anomalous values for some thermodynamic parameters (a strikingly low temperature of maximum stability, a higher than expected native-state heat capacity, and a small specific enthalpy of unfolding) could be indicative of more complex types of equilibria, such as one-state downhill folding or alternative native conformations. Moreover, double-perturbation experiments reveal some features that, in spite of the apparent linear correlation between the thermodynamic parameters, seem to be indicative of a complex conformational equilibrium in the presence of urea. In summary, the data presented here point toward the existence of a low-energy barrier between the different macrostates of hNedd4-WW4, placing it at the frontier of cooperative folding.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Calorimetry, Differential Scanning
  • Circular Dichroism
  • Endosomal Sorting Complexes Required for Transport
  • Humans
  • Hydrogen-Ion Concentration
  • Isoenzymes / chemistry
  • Nedd4 Ubiquitin Protein Ligases
  • Protein Denaturation
  • Protein Folding*
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Temperature
  • Thermodynamics*
  • Tryptophan / chemistry*
  • Ubiquitin-Protein Ligases / chemistry*

Substances

  • Endosomal Sorting Complexes Required for Transport
  • Isoenzymes
  • Tryptophan
  • Nedd4 Ubiquitin Protein Ligases
  • Nedd4 protein, human
  • Ubiquitin-Protein Ligases