Altering the association properties of insulin by amino acid replacement

Abstract

The importance of Pro^B28 and Lys^B29 on the self-association of insulin was established by systematically truncating the C terminus of the B chain. The relationship between structure and association was further explored by making numerous amino acid replacements at B²⁸ and B²⁹ Association was studied by circular dichroism, size-exclusion chromatography and ultracentrifugation. Our results show that the location of a prolyl residue at B²⁸ is critical for high-affinity self-association. Removal of Pro^B28 in a series of C-terminal truncated insulins, or amino acid replacement of Pro²⁸, greatly reduced association. The largest disruption to association was achieved by replacing Lys^B29 with Pro and varying the amino acid at B²⁸ Several of the analogs were predominantly monomers in solutions up to 3 mg/ml. These amino acid substitutions decreased association by primarily disrupting the formation of dimers. Such amino acid substitutions also substantially reduced the Zn-induced insulin hexamer formation. The formation of monomeric insulins through amino acid replacements was accompanied by conformational changes that may be the cause for decreased association. It is demonstrated that self-association of insulin can be drastically altered by substitution of one or two key amino acids.