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Title: A neutron crystallographic analysis of T{sub 6} porcine insulin at 2.1 Å resolution

Abstract

The charge balance and hydrogen-bonding network at the core of the insulin T{sub 6} hexamer have been investigated by neutron diffraction analysis at 2.1 Å resolution. Neutron diffraction data for T{sub 6} porcine insulin were collected to 2.1 Å resolution from a single crystal partly deuterated by exchange of mother liquor. A maximum-likelihood structure refinement was undertaken using the neutron data and the structure was refined to a residual of 0.179. The hydrogen-bonding network of the central core of the hexamer was observed and the charge balance between positively charged Zn ions and their surrounding structure was interpreted by considering the protonation and/or deprotonation states and interactions of HisB10, water and GluB13. The observed double conformation of GluB13 was essential to interpreting the charge balance and could be compared with the structure of a dried crystal of T{sub 6} human insulin at 100 K. Differences in the dynamic behaviour of the water molecules coordinating the upper and lower Zn ions were observed and interpreted. The hydrogen bonds in the insulin molecules, as well as those involving HisB10 and GluB13, are discussed. The hydrogen/deuterium (H/D) exchange ratios of the amide H atoms of T{sub 6} porcine insulin in crystals were obtainedmore » and showed that regions highly protected from H/D exchange are concentrated in the centre of a helical region of the B chains. From the viewpoint of soaking time versus H/D-exchange ratios, the amide H atoms can be classified into three categories.« less

Authors:
 [1];  [2];  [3];  [4];  [1];  [4];  [1]; ;  [3];  [2];  [5]
  1. Graduate School of Science and Engineering, Ibaraki University, Hitachi, Naka-Narusawa 4-12-1, Ibaraki 316-8511 (Japan)
  2. Frontier Research Center for Applied Atomic Sciences, Ibaraki University, Shirakata 162-1, Tokai, Ibaraki 319-1106 (Japan)
  3. Japan Atomic Energy Agency, Shirakata-shirane 2-4, Tokai, Ibaraki 319-1195 (Japan)
  4. Faculty of Engineering, Ibaraki University, Hitachi, Naka-Narusawa 4-12-1, Ibaraki 316-8511 (Japan)
  5. (Japan)
Publication Date:
OSTI Identifier:
22351189
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section D: Biological Crystallography; Journal Volume: 65; Journal Issue: Pt 10; Other Information: PMCID: PMC2756163; PMID: 19770501; PUBLISHER-ID: be5122; OAI: oai:pubmedcentral.nih.gov:2756163; Copyright (c) International Union of Crystallography 2009; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
Denmark
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; ATOMS; BONDING; CHAINS; DEUTERIUM; HYDROGEN; INTERACTIONS; MOLECULES; MONOCRYSTALS; NEUTRON DIFFRACTION; NEUTRONS; RESOLUTION; WATER

Citation Formats

Iwai, Wakari, Yamada, Taro, Kurihara, Kazuo, Ohnishi, Yuki, Kobayashi, Yoichiro, Tanaka, Ichiro, Takahashi, Haruyuki, Kuroki, Ryota, Tamada, Taro, Niimura, Nobuo, E-mail: niimura@mx.ibaraki.ac.jp, and Graduate School of Science and Engineering, Ibaraki University, Hitachi, Naka-Narusawa 4-12-1, Ibaraki 316-8511. A neutron crystallographic analysis of T{sub 6} porcine insulin at 2.1 Å resolution. Denmark: N. p., 2009. Web. doi:10.1107/S090744490902770X.
Iwai, Wakari, Yamada, Taro, Kurihara, Kazuo, Ohnishi, Yuki, Kobayashi, Yoichiro, Tanaka, Ichiro, Takahashi, Haruyuki, Kuroki, Ryota, Tamada, Taro, Niimura, Nobuo, E-mail: niimura@mx.ibaraki.ac.jp, & Graduate School of Science and Engineering, Ibaraki University, Hitachi, Naka-Narusawa 4-12-1, Ibaraki 316-8511. A neutron crystallographic analysis of T{sub 6} porcine insulin at 2.1 Å resolution. Denmark. doi:10.1107/S090744490902770X.
Iwai, Wakari, Yamada, Taro, Kurihara, Kazuo, Ohnishi, Yuki, Kobayashi, Yoichiro, Tanaka, Ichiro, Takahashi, Haruyuki, Kuroki, Ryota, Tamada, Taro, Niimura, Nobuo, E-mail: niimura@mx.ibaraki.ac.jp, and Graduate School of Science and Engineering, Ibaraki University, Hitachi, Naka-Narusawa 4-12-1, Ibaraki 316-8511. 2009. "A neutron crystallographic analysis of T{sub 6} porcine insulin at 2.1 Å resolution". Denmark. doi:10.1107/S090744490902770X.
@article{osti_22351189,
title = {A neutron crystallographic analysis of T{sub 6} porcine insulin at 2.1 Å resolution},
author = {Iwai, Wakari and Yamada, Taro and Kurihara, Kazuo and Ohnishi, Yuki and Kobayashi, Yoichiro and Tanaka, Ichiro and Takahashi, Haruyuki and Kuroki, Ryota and Tamada, Taro and Niimura, Nobuo, E-mail: niimura@mx.ibaraki.ac.jp and Graduate School of Science and Engineering, Ibaraki University, Hitachi, Naka-Narusawa 4-12-1, Ibaraki 316-8511},
abstractNote = {The charge balance and hydrogen-bonding network at the core of the insulin T{sub 6} hexamer have been investigated by neutron diffraction analysis at 2.1 Å resolution. Neutron diffraction data for T{sub 6} porcine insulin were collected to 2.1 Å resolution from a single crystal partly deuterated by exchange of mother liquor. A maximum-likelihood structure refinement was undertaken using the neutron data and the structure was refined to a residual of 0.179. The hydrogen-bonding network of the central core of the hexamer was observed and the charge balance between positively charged Zn ions and their surrounding structure was interpreted by considering the protonation and/or deprotonation states and interactions of HisB10, water and GluB13. The observed double conformation of GluB13 was essential to interpreting the charge balance and could be compared with the structure of a dried crystal of T{sub 6} human insulin at 100 K. Differences in the dynamic behaviour of the water molecules coordinating the upper and lower Zn ions were observed and interpreted. The hydrogen bonds in the insulin molecules, as well as those involving HisB10 and GluB13, are discussed. The hydrogen/deuterium (H/D) exchange ratios of the amide H atoms of T{sub 6} porcine insulin in crystals were obtained and showed that regions highly protected from H/D exchange are concentrated in the centre of a helical region of the B chains. From the viewpoint of soaking time versus H/D-exchange ratios, the amide H atoms can be classified into three categories.},
doi = {10.1107/S090744490902770X},
journal = {Acta Crystallographica. Section D: Biological Crystallography},
number = Pt 10,
volume = 65,
place = {Denmark},
year = 2009,
month =
}
  • Insulin is stored in pancreatic {beta}-cell as hexameric form with Zn{sup 2+} ions, while the hormonally active form is monomer. The hexamer requires the coordination of Zn{sup 2+} ions to the HisB10. In order to reveal the mechanism of the hexamerization of insulin, we investigated the Zn{sup 2+} free insulin at pD6.6 and pD9 by neutron crystallographic analyses. HisB10 is doubly protonated not only at pD6.6 but also at pD9, indicating an abnormal pK{sub a} of this histidine. It is suggested that HisB10 acts on a strong cation capture and contributes to the high stability of the hexameric form inmore » pancreas.« less
  • Acylaminoacyl peptidase from porcine liver has been crystallized. Data were collected to 3.4 Å from native crystals and a search for heavy-atom derivatives is in progress. Acylaminoacyl peptidase (also known as acylamino-acid-releasing enzyme or acylpeptide hydrolase; EC 3.4.19.1) is an unusual member of the prolyl oligopeptidase family catalysing the hydrolysis of an N-acylated peptide to an acylamino acid and a peptide with a free N-terminus. Acylaminoacyl peptidase purified from porcine liver has been crystallized in mother liquor containing 0.1 M Tris–HCl pH 7.0, 10%(w/v) polyethylene glycol 8000, 50 mM MgCl{sub 2} and 1%(w/v) CHAPS using the hanging-drop vapour-diffusion technique. Amore » full data set to 3.4 Å resolution was collected at ESRF beamline ID14-4 and space group C222 was assigned, with unit-cell parameters a = 84.8, b = 421.1, c = 212.0 Å and four molecules in the asymmetric unit.« less
  • Leydig insulin-like protein (LEY I-L) is a member of the insulin-like hormone superfamily. The LEY I-L gene (designated INSL3) is expressed exclusively in prenatal and postnatal Leydig cells. The authors report here the cloning and nucleotide sequence of porcine and human LEY I-L genes including the 5[prime] regions. Both genes consist of two exons and one intron. The organization of the LEY I-L gene is similar to that of insulin and relaxin. The transcription start site in the porcine and human LEY I-L gene is localized 13 and 14 bp upstream of the translation start site, respectively. Alignment of themore » 5[prime] flanking regions of both genes reveals that the first 107 nucleotides upstream of the transcription start site exhibit an overall sequence similarity of 80%. This conserved region contains a consensus TATAA box, a CAAT-like element (GAAT), and a consensus SP1 sequence (GGGCGG) at equivalent positions in both genes and therefore may play a role in regulation of expression of the LEY I-L gene. The porcine and human genome contains a single copy of the LEY I-L gene. By in situ hybridization, the human gene was assigned to bands p13.2-p12 of the short arm of chromosome 19. 25 refs., 6 figs.« less
  • The authors report the completion of the purification of uterine-derived growth factors (UDGF) described previously by this laboratory. During isolation, the mitogenic activity was monitored by using the human MCF-7 breast cancer cells in serum-free Ham's F12 and Dulbecco's modified Eagle's medium (1:1, v/v) containing 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 200 {mu}g/mL bovine serum albumin, and 10 {mu}g/mL human transferrin. This medium sustained growth for several days in response to a single addition of growth factor. The mitogens were shown by protein microsequencing to be DES 1 {yields} 3 to DES 1 {yields} 6 forms of insulin-like growth factor I (truncatedmore » IGF-I). An M{sub r} estimated by {sup 125}I labeling, urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography was consistent with a DES 1 {yields} 3(4) N{sup {alpha}} truncation. Immunoadsorption and radioimmunoassay confirmed immunological properties equivalent to IGF-I. Radioreceptor assays showed truncated IGF-I was functionally equivalent to recombinant IGF-I. They conclude that the major acid-stable low-M{sub r} mitogenic activities isolated from uterus are very potent forms of truncated IGF-I capable of stimulating growth of epithelial and mesenchymal cells.« less