Altering the association properties of insulin by amino acid replacement |
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Authors: | Brems David N; Alter Leila A; Beckage Michael J; Chance Ronald E; DiMarchi Richard D; Green L Kenney; Long Harlan B; Pekar Allen H; Shields James E; Frank Bruce H |
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Affiliation: | Parenteral Products Research and Development Indianapolis, IN 46285, USA
1Diabetes Research, Elt Lilly & Co. Indianapolis, IN 46285, USA |
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Abstract: | The importance of ProB28 and LysB29 on the self-associationof insulin was established by systematically truncating theC terminus of the B chain. The relationship between structureand association was further explored by making numerous aminoacid replacements at B28 and B29 Association was studied bycircular dichroism, size-exclusion chromatography and ultracentrifugation.Our results show that the location of a prolyl residue at B28is critical for high-affinity self-association. Removal of ProB28in a series of C-terminal truncated insulins, or amino acidreplacement of Pro28, greatly reduced association. The largestdisruption to association was achieved by replacing LysB29 withPro and varying the amino acid at B28 Several of the analogswere predominantly monomers in solutions up to 3 mg/ml. Theseamino acid substitutions decreased association by primarilydisrupting the formation of dimers. Such amino acid substitutionsalso substantially reduced the Zn-induced insulin hexamer formation.The formation of monomeric insulins through amino acid replacementswas accompanied by conformational changes that may be the causefor decreased association. It is demonstrated that self-associationof insulin can be drastically altered by substitution of oneor two key amino acids. |
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Keywords: | circular dichroism/ mutagenesis/ self-association/ size-exclusion/ ultracentrifugation |
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