Experimental test of the thermodynamic model of protein cooperativity using temperature-induced unfolding of a Ubq-UIM fusion protein

Biochemistry. 2010 Oct 5;49(39):8455-67. doi: 10.1021/bi101163u. Epub 2010 Sep 13.


This study describes the thermodynamic characterization of a Ubq-UIM fusion construct (Ubq-UIM), designed from the ubiquitin-UIM interaction system, to determine whether it exhibits cooperativity of folding. The Ubq-UIM fusion constructs exhibit higher stability than the core Ubq molecule, consistent with the finding that the UIM helix is docked to Ubq. Temperature-induced unfolding profiles of Ubq-UIM were monitored by DSC and far-UV and near-UV CD spectroscopies. Ubq-UIM appears to exhibit cooperative unfolding as indicated by results of global fits of a two-state model to far- and near-UV CD and DSC thermal unfolding data. The cooperativity of Ubq-UIM unfolding was further tested by the amino acid substitutions that selectively stabilize or destabilize Ubq, UIM, and/or the interface. The effects of these substitutions on the thermodynamic properties of Ubq-UIM are described well by a thermodynamic model for cooperativity in proteins. In particular, a substitution that lowered the stability of the Ubq-UIM interface indeed led to a decrease in cooperativity.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Models, Molecular
  • Molecular Sequence Data
  • Protein Interaction Domains and Motifs*
  • Protein Multimerization
  • Protein Stability
  • Protein Structure, Secondary
  • Protein Unfolding
  • Recombinant Fusion Proteins / chemistry*
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Saccharomyces cerevisiae / chemistry*
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • Temperature
  • Thermodynamics
  • Ubiquitin / chemistry*
  • Ubiquitin / genetics
  • Ubiquitin / metabolism


  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • Ubiquitin