Experimental test of the thermodynamic model of protein cooperativity using temperature-induced unfolding of a Ubq-UIM fusion protein.
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.
Patel
Mayank M
MM
Center for Biotechnology and Interdisciplinary Studies and Department of Biology, Rensselaer Polytechnic Institute, 110 8th Street, Troy, New York 12065, USA.
Sgourakis
Nikolaos G
NG
Garcia
Angel E
AE
Makhatadze
George I
GI
D000595:Amino Acid Sequence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D055550:Protein Stability; D017433:Protein Structure, Secondary; D058767:Protein Unfolding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D013816:Thermodynamics; D025801:Ubiquitin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin