Dissecting homo-heptamer thermodynamics by isothermal titration calorimetry: entropy-driven assembly of co-chaperonin protein 10.
Normally, isothermal titration calorimetry (ITC) is used to study binding reactions between two different biomolecules. Self-association processes leading to homo-oligomeric complexes have usually not been studied by ITC; instead, methods such as spectroscopy and analytical ultracentrifugation, which only provide affinity and Gibbs-free energy (i.e., K(D) and DeltaG), are employed. We here demonstrate that complete thermodynamic descriptions (i.e., K(D), DeltaG, DeltaH, and DeltaS) for self-associating systems can be obtained by ITC-dilution experiments upon proper analysis. We use this approach to probe the dissociation (and thus association) equilibrium for the heptameric co-chaperonin proteins 10 (cpn10) from Aquifex aeolicus (Aacpn10-del25) and human mitochondria (hmcpn10). We find that the midpoints for the heptamer-monomer equilibrium occur at 0.51 +/- 0.03 microM and 3.5 +/- 0.1 microM total monomer concentration (25 degrees C), for Aacpn10-del25 and hmcpn10, respectively. For both proteins, association involves endothermic enthalpy and positive entropy changes; thus, the reactions are driven by the entropy increase. This is in accord with the release of ordered water molecules and, for the thermophilic variant, a relaxation of monomer-tertiary structure when the heptamers form.
Luke
Kathryn
K
Department of Biochemistry and Cell Biology, Keck Center for Structural Computational Biology, and Department of Chemistry, Rice University, 6100 Main Street, Houston, TX 77251, USA.
Apiyo
David
D
Wittung-Stafshede
Pernilla
P
D001419:Bacteria / Q000378:metabolism; D002151:Calorimetry / Q000379:methods*; D018835:Chaperonin 10 / Q000737:chemistry* / Q000378:metabolism; D018833:Chaperonins / Q000737:chemistry; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D019277:Entropy; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D008928:Mitochondria / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors
D018835:Chaperonin 10; D011994:Recombinant Proteins; D018833:Chaperonins