title: Altering the association properties of insulin by amino acid replacement.
abstract: The importance of ProB28 and LysB29 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 ProB28 in a series of C-terminal truncated insulins, or amino acid replacement of ProB28, greatly reduced association. The largest disruption to association was achieved by replacing LysB29 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.
authors.affiliation: Parenteral Products Research and Development, Eli Lilly & Co., Indianapolis, IN 46285.
authors.forename: D N
authors.initials: DN
authors.lastname: Brems
authors.forename: L A
authors.initials: LA
authors.lastname: Alter
authors.forename: M J
authors.initials: MJ
authors.lastname: Beckage
authors.forename: R E
authors.initials: RE
authors.lastname: Chance
authors.forename: R D
authors.initials: RD
authors.lastname: DiMarchi
authors.forename: L K
authors.initials: LK
authors.lastname: Green
authors.forename: H B
authors.initials: HB
authors.lastname: Long
authors.forename: A H
authors.initials: AH
authors.lastname: Pekar
authors.forename: J E
authors.initials: JE
authors.lastname: Shields
authors.forename: B H
authors.initials: BH
authors.lastname: Frank
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives / Q000737:chemistry*; D008239:Lysine; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011392:Proline; D011487:Protein Conformation; D017510:Protein Folding; D014461:Ultracentrifugation; D015032:Zinc
chemical_list: D007328:Insulin; D011392:Proline; D015032:Zinc; D008239:Lysine