Solubility, Hydration, and Sulphate Coordination in Cubic Insulin Crystals from Esrapid ^TM Monomers Stabilized in Divalent Anionic Form of Citric Acid

Insulin’s structural adaptations have been extensively studied at neutral and basic pH; however, the effects of water and anion coordination on allosteric regions under various acidic conditions remain unexplored. Given the critical role of polar interactions in allosteric modulation, investigating structured water movements across extended pH intervals is essential for understanding solvent-mediated stabilization mechanisms in the monomer form of insulin. Structures of acid-stable cubic insulin crystals were determined in the divalent anionic form of citric acid solutions over a pH range of 2 to 6 to investigate the effects of water and anion coordination, along with charge distribution, on protein conformation. Synchrotron X-ray diffraction data were collected at resolutions ranging from 1.4 to 1.76 Å, with refined models exhibiting R -factors between 0.19 and 0.21. While the spatial arrangement of most proteins is highly conserved, ∼90% of the water coordination network and polar interactions alter local residue motion and intrinsic dynamics of the structures as the pH is changed. This is in line with previously determined structures at pH 7-11, allowing for a comprehensive structural analysis in the pH range of pH 2–11. Three key observations emerged: (i) water coordinations undergo a prominent shift toward the isoelectric point of insulin (pH 5-7), (ii) water molecules and anions function as allosteric modulators to stabilize the T-state of insulin at the pH range 2 to 6, and (iii) at extreme pH values 2 and 11, increased solubility correlates with the structural adoption of insulin’s most active form, wherein hydration within the allosteric pocket supports monomer stabilization in the T-state. Combined with the computational analyses, pH-dependent electrostatic redistributions primarily affect side-chain dynamics and local protein motion. This solvent-coupled allosteric regulation provides a mechanistic framework for solvent-mediated protein stabilization, offering a novel insight into the rational design of insulin formulations through controlled protonation and hydration strategies.