Publications (3)10.94 Total impact
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Article: Ligand Controlled Assembly of Hexamers, Dihexamers, and Linear Multihexamer Structures by the Engineered Acylated Insulin Degludec.
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ABSTRACT: Insulin degludec, an engineered acylated insulin was recently reported to form a soluble depot after subcutaneous injection with a subsequent slow release of insulin and an ultra-long glucose-lowering effect in excess of 40 hours in humans. We describe the structure, ligand binding properties and self-assemblies of insulin degludec using orthogonal structural methods. The protein fold adopted by insulin degludec is very similar to that of human insulin. Hexamers in R(6) state similar to those of human insulin are observed for insulin degludec in the presence of zinc and resorcinol. However, under conditions comparable to the pharmaceutical formulation comprising zinc and phenol insulin degludec forms finite dihexamers that are composed of hexamers in the T(3)R(3) state which interact to form an R(3)T(3)-T(3)R(3) structure. When the phenolic ligand is depleted and the solvent condition thereby mimic that of the injection site, the quaternary structure changes from dihexamers to a supramolecular structure composed of linear arrays of hundreds of hexamers in the T(3) state and an average molar mass, M0 of 59.7 x 103 kg/mole. This novel concept for self-assemblies of insulin controlled by zinc and phenol provide the basis for the slow action profile of insulin degludec. To our knowledge this report for the first time describes a tight linkage between quaternary insulin structures of hexamers, dihexamers and multihexamers and their allosteric state and its origin in the inherent propensity of the insulin hexamer to allosteric half-site reactivity.Biochemistry 12/2012; · 3.42 Impact Factor -
Article: Small Angle X-ray Scattering Based Elucidation of the Self-Association Mechanism of Human Insulin Analogue LysB29(Nεω-carboxyheptadecanoyl) des(B30).
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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. Based on small angle X-ray scattering (SAXS) supplemented by a range of biophysical methods - field flow fractionation, dynamic and multi-angle light scattering, circular dichroism, size exclusion chromatography, and crystallography - we propose a mechanism for the self-association expected to happen at subcutaneous injection of this insulin analogue. SAXS data give conclusive 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 for the mechanism of self-association for this type of insulin analogue.Biochemistry 12/2012; · 3.42 Impact Factor -
Article: Design of the novel protraction mechanism of insulin degludec, an ultra-long-acting basal insulin.
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ABSTRACT: Basal insulins with improved kinetic properties can potentially be produced using acylation by fatty acids that enable soluble, high-molecular weight complexes to form post-injection. A series of insulins, acylated at B29 with fatty acids via glutamic acid spacers, were examined to deduce the structural requirements. Self-association, molecular masses and hexameric conformations of the insulins were studied using size exclusion chromatography monitored by UV or multi-angle light scattering and dynamic light scattering, and circular dichroism spectroscopy (CDS) in environments (changing phenol and zinc concentration) simulating a pharmaceutical formulation and changes following subcutaneous injection. With depletion of phenol, insulin degludec and another fatty diacid-insulin analogue formed high molecular mass filament-like complexes, which disintegrated with depletion of zinc. CDS showed these analogues adopting stable T(3)R(3) conformation in presence of phenol and zinc, changing to T(6) with depletion of phenol. These findings suggest insulin degludec is dihexameric in pharmaceutical formulation becoming multihexameric after injection. The analogues showed weak dimeric association, indicating rapid release of monomers following hexamer disassembly. Insulins can be engineered that remain soluble but become highly self-associated after injection, slowly releasing monomers; this is critically dependent on the acylation moiety. One such analogue, insulin degludec, has therapeutic potential.Pharmaceutical Research 04/2012; 29(8):2104-14. · 4.09 Impact Factor
