INTERACTIONS OF PHENOL AND M-CRESOL IN THE INSULIN HEXAMER, AND THEIREFFECT ON THE ASSOCIATION PROPERTIES OF B28 PRO -] ASP INSULIN ANALOGS

Insulin's natural tendency to form dimers and hexamers is significantl y reduced in a mutant insulin B28 Pro --> Asp, which has been designed as a monomeric, rapid-acting hormone for therapeutic purposes. This m olecule can be induced to form zinc hexamers in the presence of small phenolic derivatives which are routinely used as antimicrobial agents in insulin preparations. Two structures of B28 Asp insulin have been d etermined from crystals grown in the presence of phenol and Nz-cresol. in these crystals, insulin exists as R-6 zinc hexamers containing a n umber of phenol or nz-cresol molecules associated with aromatic side c hains at the dimer-dimer inter-faces. At the monomer-monomer interface s, the B28 Pro --> Asp mutation leads to increased conformational flex ibility in the B chain C termini, resulting in the loss of important i ntermolecular van der Waals contacts, thus explaining the monomeric ch aracter of B28 Asp insulin. The structure of a cross-linked derivative of B28 Asp insulin, containing an Ala-Lys dipeptide linker between re sidues B30 Ala and Al Gly, has also determined. This forms an R-6 zinc hexamer containing several m-cresol molecules. Of particular interest in this structure are two nz-cresol molecules whose binding disrupted the beta-strand in one of the dimers. This observation suggests that the cross-link introduces mechanical strain on the B chain C terminus, thereby weakening the monomer-monomer interactions.