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Human insulin polymorphism upon ligand binding and pH variation: the case of 4-ethylresorcinol.

Fili S, Valmas A, Norrman M, Schluckebier G, Beckers D, Degen T, Wright J, Fitch A, Gozzo F, Giannopoulou AE, Karavassili F, Margiolaki I - IUCrJ (2015)

Bottom Line: The most representative samples were selected for synchrotron X-ray diffraction measurements, which took place at the European Synchrotron Radiation Facility (ESRF) and the Swiss Light Source (SLS).Four different crystalline polymorphs have been identified.Among these, two new phases with monoclinic symmetry have been found, which are targets for the future development of microcrystalline insulin drugs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Section of Genetics, Cell Biology and Development, Department of Biology, University of Patras , GR-26500 Patras, Greece.

ABSTRACT
This study focuses on the effects of the organic ligand 4-ethylresorcinol on the crystal structure of human insulin using powder X-ray crystallography. For this purpose, systematic crystallization experiments have been conducted in the presence of the organic ligand and zinc ions within the pH range 4.50-8.20, while observing crystallization behaviour around the isoelectric point of insulin. High-throughput crystal screening was performed using a laboratory X-ray diffraction system. The most representative samples were selected for synchrotron X-ray diffraction measurements, which took place at the European Synchrotron Radiation Facility (ESRF) and the Swiss Light Source (SLS). Four different crystalline polymorphs have been identified. Among these, two new phases with monoclinic symmetry have been found, which are targets for the future development of microcrystalline insulin drugs.

No MeSH data available.


Top, data sets for HI cocrystallized with 4-ethylresorcinol corresponding to the P21(γ) (pH 4.99–5.45) and P21(α) (pH 5.64–5.80) polymorphs. Bottom, data sets for HI cocrystallized with 4-ethylresorcinol corresponding to the C2 (pH 5.96–6.23) and P21(β) (pH 6.73–7.94) polymorphs. Data were collected on ID31 at ESRF [λ = 1.29994 (3) Å, RT].
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fig2: Top, data sets for HI cocrystallized with 4-ethylresorcinol corresponding to the P21(γ) (pH 4.99–5.45) and P21(α) (pH 5.64–5.80) polymorphs. Bottom, data sets for HI cocrystallized with 4-ethylresorcinol corresponding to the C2 (pH 5.96–6.23) and P21(β) (pH 6.73–7.94) polymorphs. Data were collected on ID31 at ESRF [λ = 1.29994 (3) Å, RT].

Mentions: High-resolution XRPD data were collected (Fig. 2 ▸) on beamline ID31 at the European Synchrotron Radiation Facility (ESRF) in Grenoble (Fitch, 2004 ▸). After loading, the capillaries were centrifuged in order to enhance the crystal packing. The capillary tubes were also spun during the measurements to avoid preferred orientation effects. Each sample was measured at several positions in order to counterbalance the radiation damage caused by the intense synchrotron beam, and several scans were collected at each position. Thus, the samples were translated by 2 mm every 4 min, exposing a fresh region of protein powder. The first scans at each position were combined in order to improve the counting statistics. The subsequent scans were only used in order to follow the evolution of the unit-cell parameters under exposure to X-ray radiation.


Human insulin polymorphism upon ligand binding and pH variation: the case of 4-ethylresorcinol.

Fili S, Valmas A, Norrman M, Schluckebier G, Beckers D, Degen T, Wright J, Fitch A, Gozzo F, Giannopoulou AE, Karavassili F, Margiolaki I - IUCrJ (2015)

Top, data sets for HI cocrystallized with 4-ethylresorcinol corresponding to the P21(γ) (pH 4.99–5.45) and P21(α) (pH 5.64–5.80) polymorphs. Bottom, data sets for HI cocrystallized with 4-ethylresorcinol corresponding to the C2 (pH 5.96–6.23) and P21(β) (pH 6.73–7.94) polymorphs. Data were collected on ID31 at ESRF [λ = 1.29994 (3) Å, RT].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4547821&req=5

fig2: Top, data sets for HI cocrystallized with 4-ethylresorcinol corresponding to the P21(γ) (pH 4.99–5.45) and P21(α) (pH 5.64–5.80) polymorphs. Bottom, data sets for HI cocrystallized with 4-ethylresorcinol corresponding to the C2 (pH 5.96–6.23) and P21(β) (pH 6.73–7.94) polymorphs. Data were collected on ID31 at ESRF [λ = 1.29994 (3) Å, RT].
Mentions: High-resolution XRPD data were collected (Fig. 2 ▸) on beamline ID31 at the European Synchrotron Radiation Facility (ESRF) in Grenoble (Fitch, 2004 ▸). After loading, the capillaries were centrifuged in order to enhance the crystal packing. The capillary tubes were also spun during the measurements to avoid preferred orientation effects. Each sample was measured at several positions in order to counterbalance the radiation damage caused by the intense synchrotron beam, and several scans were collected at each position. Thus, the samples were translated by 2 mm every 4 min, exposing a fresh region of protein powder. The first scans at each position were combined in order to improve the counting statistics. The subsequent scans were only used in order to follow the evolution of the unit-cell parameters under exposure to X-ray radiation.

Bottom Line: The most representative samples were selected for synchrotron X-ray diffraction measurements, which took place at the European Synchrotron Radiation Facility (ESRF) and the Swiss Light Source (SLS).Four different crystalline polymorphs have been identified.Among these, two new phases with monoclinic symmetry have been found, which are targets for the future development of microcrystalline insulin drugs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Section of Genetics, Cell Biology and Development, Department of Biology, University of Patras , GR-26500 Patras, Greece.

ABSTRACT
This study focuses on the effects of the organic ligand 4-ethylresorcinol on the crystal structure of human insulin using powder X-ray crystallography. For this purpose, systematic crystallization experiments have been conducted in the presence of the organic ligand and zinc ions within the pH range 4.50-8.20, while observing crystallization behaviour around the isoelectric point of insulin. High-throughput crystal screening was performed using a laboratory X-ray diffraction system. The most representative samples were selected for synchrotron X-ray diffraction measurements, which took place at the European Synchrotron Radiation Facility (ESRF) and the Swiss Light Source (SLS). Four different crystalline polymorphs have been identified. Among these, two new phases with monoclinic symmetry have been found, which are targets for the future development of microcrystalline insulin drugs.

No MeSH data available.