title: Small angle X-ray scattering-based elucidation of the self-association mechanism of human insulin analogue lys(B29)(N(ε)ω-carboxyheptadecanoyl) des(B30).
abstract: Lys(B29)(N(ε)ω-carboxyheptadecanoyl) des(B30) human insulin is an insulin analogue belonging to a class of analogues designed to form soluble depots in subcutis by self-association, aiming at a protracted action. On the basis of small angle X-ray scattering (SAXS) supplemented by a range of biophysical and structural methods (field flow fractionation, dynamic and multiangle light scattering, circular dichroism, size exclusion chromatography, and crystallography), we propose a mechanism for the self-association expected to occur upon subcutaneous injection of this insulin analogue. SAXS data provide evidence of the in solution structure of the self-associated oligomer, which is a long straight rod composed of "tense" state insulin hexamers (T(6)-hexamers) as the smallest repeating unit. The smallest oligomer building block in the process is a T(6)T(6)-dihexamer. This tense dihexamer is formed by the allosteric change of the initial equilibrium between a proposed "relaxed" state R(6)-hexamer and an R(3)T(3)T(3)R(3)-dihexamer. The allosteric change from relaxed to tense is triggered by removal of phenol, mimicking subcutaneous injection. The data hence provide the first unequivocal evidence of the mechanism of self-association for this type of insulin analogue.
authors.affiliation: Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark.
authors.forename: Malene Hillerup
authors.initials: MH
authors.lastname: Jensen
authors.forename: Per-Olof
authors.initials: PO
authors.lastname: Wahlund
authors.forename: Katrine Nørgaard
authors.initials: KN
authors.lastname: Toft
authors.forename: Jes Kristian
authors.initials: JK
authors.lastname: Jacobsen
authors.forename: Dorte Bjerre
authors.initials: DB
authors.lastname: Steensgaard
authors.forename: Marco
authors.initials: M
authors.lastname: van de Weert
authors.forename: Svend
authors.initials: S
authors.lastname: Havelund
authors.forename: Bente
authors.initials: B
authors.lastname: Vestergaard
mesh_terms: D018360:Crystallography, X-Ray; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives* / Q000737:chemistry; D008958:Models, Molecular; D055503:Protein Multimerization; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction
chemical_list: D007328:Insulin