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Dissecting Homo-Heptamer Thermodynamics by Isothermal Titration Calorimetry: Entropy-Driven Assembly of Co-Chaperonin Protein 10

Kathryn Luke ^*, David Apiyo ^* and Pernilla Wittung-Stafshede ^*  

^* Department of Biochemistry and Cell Biology, Keck Center for Structural Computational Biology, and Department of Chemistry, Rice University, Houston, Texas

Correspondence: Address reprint requests to P. Wittung-Stafshede, Dept. of Biochemistry and Cell Biology, MS-140, Rice University, 6100 Main St., Houston, TX 77251. Tel.: 713-348-4076; E-mail: pernilla{at}rice.edu.

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 G), are employed. We here demonstrate that complete thermodynamic descriptions (i.e., K_D, G, H, and S) 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 µM and 3.5 ± 0.1 µM total monomer concentration (25°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.

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