Hydration and structure—the two sides of the insulin aggregation process

Abstract

Aggregation of bovine insulin under monomer, and dimer-promoting conditions has been probed by FT-IR spectroscopy and DSC/PPC calorimetry. This approach enabled linking of characteristic aggregation-hallmarking conformational events (such as partial unfolding, and subsequent refolding into aggregated β-strands) to different stages of protein hydration. Aggregation of insulin in 20% acetic acid occurs at a markedly lower temperature than in pure water and is further enhanced in the presence of charge-shielding ions (0.1 M NaCl). While acetic acid makes the protein molecule more permissive to the solvent penetration (seen as the enhanced H/D-exchange), NaCl does not have this effect. The calorimetric data reveals that, while the aggregation itself is exothermic, in the absence of the co-solvent it is preceded by an endothermic transition. In either case, the negative heat capacity changes suggest a substantial reduction in the number of water–protein contacts upon aggregation. This has been supported by pressure perturbation calorimetry, which showed a simultaneous release of water molecules bound to the protein. The aggregation of insulin appears to be driven mostly by hydrophobic interactions and, as such, involves a rearrangement of hydrophobic side-chain amino acid residues that leads to the overall reduction in number of unfavorable protein–water contacts. The study points to the intricate nature of the acetic acid effect on insulin aggregation, which is likely to involve changing the structure of the solvating water, and direct binding to the protein.