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Dissecting homo-heptamer thermodynamics by isothermal titration calorimetry: entropy-driven assembly of co-chaperonin protein 10.Luke K, Apiyo D, Wittung-Stafshede P 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. 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. Published 28 October 2005 in Biophys J, 89(5): 3332-6.
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