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

X-ray analysis of insulin crystals has revealed the nature of the surf aces involved in its assembly to dimers and hexamers. The protein cont acts between monomers are well defined but can vary. Contacts between dimers in the hexamer are generally looser and can change remarkably i n their structure, particularly by the existence of extended or helica l conformations at the N terminus of the B chain. The assembly of insu lin to hexamers is associated with the hormone's slow absorption by ti ssue, 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 absorpt ion characteristics. The most effective mechanism to favour the monome ric state was the introduction of carboxylic acids which generated ele ctrostatic repulsion in the dimer and hexamer species. Some of the mut ated insulins have been crystallized and their structures determined, revealing the structural basis of their assembly properties. In the pr esence of chloride or phenol (and related molecules) the otherwise ext ended structure of residue B1-B8 forms an alpha helix, packing against the adjacent dimer. This provides additional sites for zinc at the di mer-dimer interfaces, and also can provide a binding site for phenol a nd related molecules. The surfaces in this cavity provide a template f or modelling in other ligands.