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Publications (6)21.44 Total impact

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    ABSTRACT: Insulin is a key hormone controlling glucose homeostasis. All known vertebrate insulin analogues have a classical structure with three 100% conserved disulfide bonds that are essential for structural stability and thus the function of insulin. It might be hypothesized that an additional disulfide bond may enhance insulin structural stability which would be highly desirable in a pharmaceutical use. To address this hypothesis, we designed insulin with an additional interchain disulfide bond in positions A10/B4 based on Cα-Cα distances, solvent exposure and side-chain orientation in human insulin structure. This insulin analogue had increased affinity for the insulin receptor and apparently augmented glucodynamic potency in a normal rat model compared to human insulin. Addition of the disulfide bond also resulted in a 34.6 °C increase in melting temperature and prevented insulin fibril formation under high physical stress even though the C-terminus of the B-chain thought to be directly involved in fibril formation was not directly stabilized. Importantly, this analogue was capable of forming hexamer upon Zn addition as typical for wild-type insulin and its crystal structure showed only minor deviations from the classical insulin structure. Furthermore, the additional disulfide bond prevented this insulin analogue from adopting the R-state conformation and thus showing that the R-state conformation is not a prerequisite for binding to insulin receptor as previously suggested. In summary, this is the first example of an insulin analogue featuring a fourth disulfide bond with increased structural stability and retained function. Proteins 2012. © 2012 Wiley Periodicals, Inc.
    Protein Science 12/2012; · 2.74 Impact Factor
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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; · 3.38 Impact Factor
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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; · 3.38 Impact Factor
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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. · 4.74 Impact Factor
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    ABSTRACT: We have explored the adsorption of zinc-free human insulin on the three low-index single-crystalline Au(111)-, Au(100)- and Au(110)-surfaces in aqueous buffer (KH(2)PO(4), pH 5) by a combination of electrochemical scanning tunnelling microscopy (in situ STM) at single-molecule resolution and linear sweep, LSV, cyclic, CV, and square wave (SQWV) voltammetry.Multifarious electrochemical patterns were observed. Most attention was given to reductive desorption caused by insulin binding to the Au-surfaces via up to three disulfide groups per insulin monomer, presumably converted to single Au-S links. SQWV suggested the Au-S bond strength order Au(111) > Au(110) > Au(100) based on the reductive desorption potentials. The voltammetric diversity was paralleled by different in situ STM insulin adsorption modes on the three surfaces. Single-molecule resolution was achieved in all cases. The coverage followed the order Au(110) > Au(100) > Au(111) and differs from the reductive desorption order that records the Au-S bonding element. Evenly distributed single molecules were scattered over large Au(111)-terraces, with intriguing molecular arrays disclosed near the terrace edges. In comparison, high-density molecular scale structures were observed both over the terraces and across terrace edges on Au(100). Larger rectangular structures also appeared (8-12% coverage). These are Au-islands from the lift of the reconstruction. Notably, 10 x 10 nm(2) patches of highly ordered much smaller structures, possibly from insulin decomposition emerged sporadically within the dense insulin adlayer. Insulin adsorbed in highest coverage on the Au(110) and followed the directional surface topology with insulin molecules aligned in the Au(110)-surface grooves, occasionally "spilling over" and merging into larger structures.Adsorption, Au-S binding, and insulin unfolding are all parts of insulin surface behaviour and reflected in both voltammetry and in situ STM. In spite of these complications, the data show that molecular scale resolution has been achieved and offer other perspectives of insulin surface science such as single-molecule mapping of the insulin monomer/dimer-hexamer interconversion.
    Physical Chemistry Chemical Physics 09/2010; 12(34):9999-10011. · 3.83 Impact Factor
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    ABSTRACT: Through binding to and signaling via the insulin receptor (IR), insulin is involved in multiple effects on growth and metabolism. The current model for the insulin-IR binding process is one of a biphasic reaction. It is thought that the insulin peptide possesses two binding interfaces (sites 1 and 2), which allow it to bridge the two alpha-subunits of the insulin receptor during the biphasic binding reaction. The sequential order of the binding events involving sites 1 and 2, as well as the molecular interactions corresponding to the fast and slow binding events, is still unknown. In this study we examined the series of events that occur during the binding process with the help of three insulin analogues: insulin, an analogue mutated in site 2 (B17A insulin), and an analogue in which part of site 1 was deleted (Des A1-4 insulin), both with and without a fluorescent probe attached. The binding properties of these analogues were tested using two soluble Midi IR constructs representing the two naturally occurring isoforms of the IR, Midi IR-A and Midi IR-B. Our results showed that in the initial events leading to Midi IR-insulin complex formation, insulin site 2 binds to the IR in a very fast binding event. Subsequent to this initial fast phase, a slower rate-limiting phase occurs, consistent with a conformational change in the insulin-IR complex, which forms the final high-affinity complex. The terminal residues A1-A4 of the insulin A-chain are shown to be important for the slow binding phase, as insulin lacking these amino acids is unable to induce a conformational change of IR and has a severely impaired binding affinity. Moreover, differences in the second phase of the binding process involving insulin site 1 between the IR-A and IR-B isoforms suggest that the additional amino acids encoded by exon 11 in the IR-B isoform influence the binding process.
    Biochemistry 07/2010; 49(29):6234-46. · 3.38 Impact Factor