INSULIN ASSEMBLY - ITS MODIFICATION BY PROTEIN ENGINEERING AND LIGAND-BINDING

X-ray analysis of insulin crystals has revealed the nature of the surfaces involved in its assembly to dimers and hexamers. The protein contacts between monomers are well defined but can vary. Contacts between dimers in the hexamer are generally looser and can change remarkably in their structure, particularly by the existence of extended or helical conformations at the N terminus of the B chain. The assembly of insulin to hexamers is associated with the hormone's slow absorption by tissue, in the diabetic this can lead to inappropriate insulin levels in the blood. Experiments to improve insulin absorption at the injection site have been based on constructing 'monomeric' insulins by protein engineering. These have led to stable monomers with more rapid absorption characteristics. The most effective mechanism to favour the monomeric state was the introduction of carboxylic acids which generated electrostatic repulsion in the dimer and hexamer species. Some of the mutated insulins have been crystallized and their structures determined, revealing the structural basis of their assembly properties. In the presence of chloride or phenol (and related molecules) the otherwise extended structure of residue B1-B8 forms an alpha helix, packing against the adjacent dimer. This provides additional sites for zinc at the dimer-dimer interfaces, and also can provide a binding site for phenol and related molecules. The surfaces in this cavity provide a template for modelling in other ligands.