Small Angle X‑ray Scattering-Based Elucidation of the Self-Association Mechanism of Human Insulin Analogue LysB29(Nεω‑carboxyheptadecanoyl) des(B30)

LysB29(Nεω-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 (T6-hexamers) as the smallest repeating unit. The smallest oligomer building block in the process is a T6T6-dihexamer. This tense dihexamer is formed by the allosteric change of the initial equilibrium between a proposed “relaxed” state R6-hexamer and an R3T3T3R3-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.