Small angle X-ray scattering-based elucidation of the self-association mechanism of human insulin analogue lys(B29)(N(e)¿-carboxyheptadecanoyl) des(B30)

Malene Hillerup Jensen; Per-Olof Wahlund; Katrine Nørgaard Toft; Jes Kristian Jacobsen; Dorte Bjerre Steensgaard; Marco van de Weert; Svend Havelund; Bente Vestergaard

doi:10.1021/bi3008615

Small angle X-ray scattering-based elucidation of the self-association mechanism of human insulin analogue lys(B29)(N(e)¿-carboxyheptadecanoyl) des(B30)

Malene Hillerup Jensen, Per-Olof Wahlund, Katrine Nørgaard Toft, Jes Kristian Jacobsen, Dorte Bjerre Steensgaard, Marco van de Weert, Svend Havelund, Bente Vestergaard

13 Citations (Scopus)

Abstract

Lys^B29(N^εω-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 R₃T ₃T₃R₃-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.

Original language	English
Journal	Biochemistry
Volume	52
Issue number	2
Pages (from-to)	282-94
Number of pages	13
ISSN	0006-2960
DOIs	https://doi.org/10.1021/bi3008615
Publication status	Published - 15 Jan 2013

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Small angle X-ray scattering-based elucidation of the self-association mechanism of human insulin analogue lys(B29)(N(e)¿-carboxyheptadecanoyl) des(B30). / Jensen, Malene Hillerup; Wahlund, Per-Olof; Toft, Katrine Nørgaard et al.

In: Biochemistry, Vol. 52, No. 2, 15.01.2013, p. 282-94.

Research output: Contribution to journal › Journal article › Research › peer-review

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title = "Small angle X-ray scattering-based elucidation of the self-association mechanism of human insulin analogue lys(B29)(N(e)¿-carboxyheptadecanoyl) des(B30)",

abstract = "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 R3T 3T3R3-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.",

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AU - Wahlund, Per-Olof

AU - Toft, Katrine Nørgaard

AU - Jacobsen, Jes Kristian

AU - Steensgaard, Dorte Bjerre

AU - van de Weert, Marco

AU - Havelund, Svend

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N2 - 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 R3T 3T3R3-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.

AB - 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 R3T 3T3R3-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.

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Small angle X-ray scattering-based elucidation of the self-association mechanism of human insulin analogue lys(B29)(N(e)¿-carboxyheptadecanoyl) des(B30)

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