Insulin Polymorphism Crystallographic Characterization of Insulin Microcrystals

Mathias Norrman

Research output: ThesisDoctoral Thesis (compilation)

Abstract

Insulin is a protein needed for the uptake of glucose from the circulating blood. In absence of sufficiently high insulin levels in the body or when the cells have a reduced sensitivity for insulin a disease state referred to as diabetes occurs. Treatment of diabetes generally requires daily injections of exogenous insulin. Many of the pharmaceutical formulations consist of insulin in a crystalline state. After injection, the crystals start to dissolve and the insulin molecules diffuse into the bloodstream. Crystal size, morphology and crystal packing affect the action profile of the formulation. Other factors with impact on the duration include additives and ligands such as zinc and phenolic molecules.

A careful characterization of the microcrystals is important both from a research perspective but also for regulatory reasons. Within this project, X-ray crystallography was used to characterize microcrystals of insulin. The small size of the crystals makes visual interpretation difficult, and determination of crystal form based on crystal morphology is sometimes not possible. General single crystal X-ray analysis is of limited use for characterization of the microcrystals since the crystals are too small. X-ray powder diffraction was therefore utilized. Several suspensions of insulin microcrystals were characterized by this method. Insulin is a polymorphic protein that can be crystallized in a number of different crystal forms and space groups. It was shown that the different crystal forms had specific X-ray powder patterns. The patterns could thus serve as fingerprints for certain crystal forms. Both pharmaceutical formulations and crystalline suspension from research activities could be characterized. Polymorphism within samples could be detected and even two new crystal forms of insulin were identified. For efficient analysis of the powder patterns a multivariate analysis method was used, principal component analysis (PCA). This facilitated analysis and visualization of the data considerably.

One of the new crystal forms was subsequently structurally determined by single crystal analysis after modification of the crystallization conditions to promote growth of larger crystals. The space group was orthorhombic C2221. The asymmetric unit contained three hexamers with a novel crystal packing between hexamers. By increasing the pH by ~0.5 pH units to 7.0 a second new crystal form was found (monoclinic C2). The major difference was a fewer number of the novel crystal packing interactions between the hexamers in this crystal. The packing contact involves two tyrosine-tyrosine interactions and a tight glutamate-glutamate interaction of 2.4 Å. When pH was increased further (above 7.0) the dominant crystal form was the previously well characterized monoclinic P21, with no crystal packing interactions of this kind.

The powder diffraction methods utilized in this project was useful for determination of crystal form of the microcrystals. As a complement, it was shown that the microcrystals could be used to solve the structure by using a microfocused X-ray beam at a designated beamline. The structure of human insulin was solved at a resolution of 2.2 Å, from orthorhombic crystals in space group P212121. The human insulin was co-crystallized with the peptide protamine consisting mainly of arginine residues. Such crystals have long been used for the treatment of diabetes and are referred to as NPH crystals (neutral protamine Hagedorn). Due to crystallographic disorder of the peptide, the protamine could however not be identified in the electron density map. The disorder suggests that the insulin-protamine interaction is unspecific and that the primary function of the protamine is to balance the overall charge of insulin.
Original languageEnglish
QualificationDoctor
Awarding Institution
  • Biochemistry and Structural Biology
Supervisors/Advisors
  • Al-Karadaghi, Salam, Supervisor
Award date2007 Feb 2
Publisher
ISBN (Print)978-91-7422-144-2
Publication statusPublished - 2007

Bibliographical note

Defence details

Date: 2007-02-02
Time: 13:15
Place: Kemicentrum, sal D Lund

External reviewer(s)

Name: Hilgenfeld, Rolf
Title: Professor
Affiliation: Inst. of Biochemistry, University of Lübeck

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Subject classification (UKÄ)

  • Biological Sciences

Keywords

  • Strukturkemi
  • Structure chemistry
  • biomatematik
  • protein crystallography
  • X-ray powder diffraction
  • Bioinformatics
  • medical informatics
  • microcrystals
  • Bioinformatik
  • Molekylär biofysik
  • Molecular biophysics
  • biomathematics biometrics
  • medicinsk informatik
  • insulin

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