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Hierarchical Modeling of Phenolic Ligand Binding to 2Zn−Insulin Hexamers
Phenolic ligands, e.g., phenol and m-cresol, bind to 2Zn(II)−insulin hexamers and induce a conformational change at the N-terminus of the B-chain for each monomer. The binding of these phenolic ligands to 2Zn(II)−insulin hexamers has been studied by isothermal titrating calorimetry (ITC). The bindin...
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Published in: | Biochemistry (Easton) 1996-04, Vol.35 (17), p.5366-5378 |
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Main Authors: | , , , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | Phenolic ligands, e.g., phenol and m-cresol, bind to 2Zn(II)−insulin hexamers and induce a conformational change at the N-terminus of the B-chain for each monomer. The binding of these phenolic ligands to 2Zn(II)−insulin hexamers has been studied by isothermal titrating calorimetry (ITC). The binding isotherms were modeled and thermodynamic parameters were quantified using a novel, flexible algorithm that permitted the development of a hierarchical series of physical models. With the insulin hexamer represented as a dimer of trimers, the modeling demonstrated that ligand binding is highly cooperative in nature, both intra- and inter-trimer. The isotropic inter-trimer cooperativity was dominant and negative in every system studied, with initial binding constants typically an order of magnitude greater for the binding of ligands to the first trimer relative to the second. The inter-trimer cooperativity estimated from the modeling of solution calorimetry data is consistent with a T6 ↔ T3R3 ↔ R6 equilibrium first proposed from crystallographic investigations. Intra-trimer cooperativity was present only in the enthalpy coefficient space, not in the equilibrium coefficient space, and therefore, less of a factor. The order of binding affinity for the ligands studied is resorcinol ≫ phenol ≥ m-cresol as determined from their overall free energies of binding to the 2Zn(II)−insulin hexamer (−26.6, −23.4, and −23.4 kcal/mol, respectively) and their intrinsic binding constants (8780, 5040, and 3370 L/mol, respectively) at 14 °C. The temperature dependence of phenol binding to 2Zn(II)−insulin hexamer was modeled. Increasing temperature decreased the magnitude of both the intrinsic binding constant and the inter-trimer cooperativity. The second phase of the ITC binding profile was also found to be highly temperature dependent. At lower temperatures the second phase is endothermic but gradually decreases with increasing temperature and subsequently becomes exothermic. This effect is attributed to loss of water from the hydration shell of the insulin hexamer with increasing temperature and consequently reduces the entropic contributions to the T ↔ R transition in the phenol/2Zn(II)−insulin hexamer system. |
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ISSN: | 0006-2960 1520-4995 |
DOI: | 10.1021/bi9600557 |