Thermodynamics of Formation of the Insulin Hexamer: Metal-Stabilized Proton-Coupled Assembly of Quaternary Structure

The thermodynamics of formation of the insulin hexamer, which is stabilized by two Zn²⁺ ions, were quantified by isothermal titration calorimetry (ITC). Because the insulin monomer is unstable to aggregation (fibrillation) during ITC measurements, an original method involving EDTA chelation of Zn²⁺ from the hexamer was employed. The two metal ions are chelated sequentially, reflecting stepwise Zn²⁺ binding and stabilization of the quaternary structure. Analysis of the ITC data reveals that two to three H⁺ bind to the hexamer upon its formation at pH 7.4, which is both enthalpically and entropically favored. The former is due to Zn²⁺ coordination to His residues from three subunits, and the latter is associated with desolvation that accompanies the protonation and the packing of the subunits in the hexamer