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Title: Crystallization of Spätzle, a cystine-knot protein involved in embryonic development and innate immunity in Drosophila melanogaster

Abstract

Crystallization of the cystine-knot protein Spätzle occurred following serendipitous limited degradation of the pro-Spätzle propeptide during the crystallization experiment. The Spätzle protein is involved in both the definition of the dorsal–ventral axis during embryonic development and in the adult innate immune response. The disulfide-linked dimeric cystine-knot protein has been expressed as a proprotein in inclusion bodies in Escherichia coli and refolded in vitro by rapid dilution. Initial orthorhombic crystals that diffracted to 7 Å resolution were obtained after three months by the sitting-drop vapour-diffusion method. Optimization of the crystallization conditions resulted in orthorhombic crystals (space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 53.0, b = 59.2, c = 62.5 Å) that diffracted to 2.8 Å resolution in-house. The small volume of the asymmetric unit indicated that it was not possible for the crystals to contain the complete pro-Spätzle dimer. Mass spectrometry, N-terminal sequencing and Western-blot analysis revealed that the crystals contained the C-terminal disulfide-linked cystine-knot dimer. Comparison of various crystallization experiments indicated that degradation of the N-terminal prodomain was dependent on the buffer conditions.

Authors:
;  [1];  [2]
  1. Institut für Biochemie und Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Abteilung Physikalische Biotechnologie, Kurt-Mothes-Strasse 3, 06120 Halle (Saale) (Germany)
  2. Max-Planck-Institut für Proteinfaltung, Abteilung Massenspektrometrie, Kurt-Mothes-Strasse 3, 06120 Halle (Saale) (Germany)
Publication Date:
OSTI Identifier:
22360612
Resource Type:
Journal Article
Journal Name:
Acta Crystallographica. Section F
Additional Journal Information:
Journal Volume: 64; Journal Issue: Pt 8; Other Information: PMCID: PMC2494967; PMID: 18678937; PUBLISHER-ID: gj5042; OAI: oai:pubmedcentral.nih.gov:2494967; Copyright (c) International Union of Crystallography 2008; Country of input: International Atomic Energy Agency (IAEA); Journal ID: ISSN 1744-3091
Country of Publication:
United Kingdom
Language:
English
Subject:
75 CONDENSED MATTER PHYSICS, SUPERCONDUCTIVITY AND SUPERFLUIDITY; BUFFERS; CRYSTALLIZATION; CRYSTALS; DIFFUSION; DILUTION; DIMERS; ESCHERICHIA COLI; IN VITRO; INCLUSIONS; MASS SPECTROSCOPY; OPTIMIZATION; RESOLUTION; SPACE GROUPS

Citation Formats

Hoffmann, Anita, Neumann, Piotr, Schierhorn, Angelika, Stubbs, Milton T., E-mail: stubbs@biochemtech.uni-halle.de, and Mitteldeutsches Zentrum für Struktur und Dynamik der Proteine. Crystallization of Spätzle, a cystine-knot protein involved in embryonic development and innate immunity in Drosophila melanogaster. United Kingdom: N. p., 2008. Web. doi:10.1107/S1744309108018812.
Hoffmann, Anita, Neumann, Piotr, Schierhorn, Angelika, Stubbs, Milton T., E-mail: stubbs@biochemtech.uni-halle.de, & Mitteldeutsches Zentrum für Struktur und Dynamik der Proteine. Crystallization of Spätzle, a cystine-knot protein involved in embryonic development and innate immunity in Drosophila melanogaster. United Kingdom. https://doi.org/10.1107/S1744309108018812
Hoffmann, Anita, Neumann, Piotr, Schierhorn, Angelika, Stubbs, Milton T., E-mail: stubbs@biochemtech.uni-halle.de, and Mitteldeutsches Zentrum für Struktur und Dynamik der Proteine. 2008. "Crystallization of Spätzle, a cystine-knot protein involved in embryonic development and innate immunity in Drosophila melanogaster". United Kingdom. https://doi.org/10.1107/S1744309108018812.
@article{osti_22360612,
title = {Crystallization of Spätzle, a cystine-knot protein involved in embryonic development and innate immunity in Drosophila melanogaster},
author = {Hoffmann, Anita and Neumann, Piotr and Schierhorn, Angelika and Stubbs, Milton T., E-mail: stubbs@biochemtech.uni-halle.de and Mitteldeutsches Zentrum für Struktur und Dynamik der Proteine},
abstractNote = {Crystallization of the cystine-knot protein Spätzle occurred following serendipitous limited degradation of the pro-Spätzle propeptide during the crystallization experiment. The Spätzle protein is involved in both the definition of the dorsal–ventral axis during embryonic development and in the adult innate immune response. The disulfide-linked dimeric cystine-knot protein has been expressed as a proprotein in inclusion bodies in Escherichia coli and refolded in vitro by rapid dilution. Initial orthorhombic crystals that diffracted to 7 Å resolution were obtained after three months by the sitting-drop vapour-diffusion method. Optimization of the crystallization conditions resulted in orthorhombic crystals (space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 53.0, b = 59.2, c = 62.5 Å) that diffracted to 2.8 Å resolution in-house. The small volume of the asymmetric unit indicated that it was not possible for the crystals to contain the complete pro-Spätzle dimer. Mass spectrometry, N-terminal sequencing and Western-blot analysis revealed that the crystals contained the C-terminal disulfide-linked cystine-knot dimer. Comparison of various crystallization experiments indicated that degradation of the N-terminal prodomain was dependent on the buffer conditions.},
doi = {10.1107/S1744309108018812},
url = {https://www.osti.gov/biblio/22360612}, journal = {Acta Crystallographica. Section F},
issn = {1744-3091},
number = Pt 8,
volume = 64,
place = {United Kingdom},
year = {Fri Aug 01 00:00:00 EDT 2008},
month = {Fri Aug 01 00:00:00 EDT 2008}
}