Forskning ved Københavns Universitet - Københavns Universitet

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Insulin Self-Assembly and Carbohydrate Labeling

Publikation: Bog/antologi/afhandling/rapportPh.d.-afhandlingForskning

  • Mads Østergaard
The work described in this thesis has focused on the reversible, higher-ordered, hierarchical selfassembly of insulin analogs modified by metal ion-coordinating ligands. The dynamic selfassemblyof insulin shows potential in improving diabetes treatment. Human insulin has been site-selectively modified with a series of metal binding organic molecules, and the subsequent self-assembly of the modified insulins, in the presence of metal ions, has been studied. The main study has been on carbohydrate-modified insulin. Oligogalacturonic acids, which are capable of forming dimers and multimers in the presence of Ca2+, have been isolated and conjugated to insulin. This modification was achieved through formation of an oxime linkage after site-selective introduction of an aminooxy moiety into insulin under pH control. Studies on the self-assembly of the carbohydrate insulins argued that the insulins were capable of undergoing hexamer formation after modification. For insulins carrying long oligogalacturonic acid chains, further hierarchical self-assembly of the insulin hexamers occurred through dimerization of the oligogalacturonic acid ligands after addition of Ca2+. Structural changes induced by Ca2+ were studied by circular dichroism spectroscopy, while size and shape of selfassemblies were studied using light scattering and high-resolution microscopy techniques. It was found that self-assembly could be reversed by addition of a metal chelating agent, which sequesters Ca2+ ions. A binding assay revealed that the carbohydrate-insulins maintained affinity towards the insulin receptor, confirming their potential use in the treatment of diabetes. The chemistry and nucleophilic catalysis of carbohydrate oxime formation has been thoroughly studied. Reactions of D-glucose, D-glucuronic acid, and 2-acetamido-2-deoxy-D-glucose with various α-nucleophiles under catalysis by a series of anilinic nucleophiles at different pH values have been carried out and analyzed by high-performance liquid chromatography. The study provides a detailed overview of reaction kinetics, and the exceptional catalysis by pphenylenediamine at neutral pH was investigated by nuclear magnetic resonance spectroscopy and molecular modeling. Human insulin was also modified by bipyridine, a metal binding ligand, which binds Fe2+ in the formation of a chiral triplex. The metal complex absorbs visible light, giving rise to a magenta color of solutions containing insulin analogs self-assembled through the coordination of Fe2+. Combined with the hexameric self-assembly of insulin, stabilized by Zn2+ ions, the Fe2+-induced bipyridine triplex formation gave rise to a trimer of insulin hexamers, or an insulin 18-mer, as shown by X-ray scattering studies. Self-assemblies in the presence of metal ions were further characterized using high-resolution microscopy, and the reversibility of self-assemblies was documented through chromatographic and spectroscopic studies. In vivo studies found that the modified insulin was capable of reducing the blood glucose level of rats. Finally, terpyridine, a metal binding ligand capable of forming a duplex through binding of divalent metal ions, has been introduced into insulin. As for bipyridine, terpyridine binding of Fe2+ yields a complex of magenta color. The self-association of the terpyridine-insulin analog was studied mainly by light spectroscopy. Protein crystallographic studies were pursued for this insulin analog, resulting in the formation of cubic insulin crystals. In the crystal, disorder led to an unresolvable ligand site, although the protein segment of the molecule could be revealed. Crystals formed in the presence of Fe2+ were magenta in color, documenting terpyridine duplex formation inside the crystal. At the ligand site of the crystal, a three-fold symmetry axis was found. Eu3+, which is capable of coordinating a terpyridine triplex, facilitated formation of cubic crystals much more rapidly than Fe2+ ions, but did not provide structure at the ligand site.
OriginalsprogEngelsk
ForlagDepartment of Chemistry, Faculty of Science, University of Copenhagen
StatusUdgivet - 2017

ID: 215042674