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Purification of FarnesylatedhGBP1 and Characterization of Its Polymerization and Membrane Binding

  • Linda Sistemich
  • Christian HerrmannEmail author
Protocol
  • 81 Downloads
Part of the Methods in Molecular Biology book series (MIMB, volume 2159)

Abstract

The human guanylate-binding protein 1 (hGBP1) is the best characterized isoform of the seven human GBPs belonging to the superfamily of dynamin-like proteins (DLPs). As known for other DLPs, hGBP1 also exhibits antiviral and antimicrobial activity within the cell. hGBP 1, like hGBPs 2 and 5, carries a CAAX motive at the C-terminus leading to isoprenylation in the living cells. The attachment of a farnesyl anchor and its unique GTPase cycle provides hGBP1 the ability of a nucleotide- stimulated polymerization and membrane binding. In this chapter, we want to show how to prepare farnesylated hGBP1 (hGBP1fn) by bacterial synthesis and by enzymatic modification, respectively, and how to purify the non-farnesylated, as well as the farnesylated hGBP1, by chromatographic procedures. Furthermore, we want to demonstrate how to investigate the special features of polymerization by a UV-absorption-based turbidity assay and the binding to artificial membranes by means of fluorescence energy transfer.

Key words

Large GTPases GBP Farnesylation Membrane binding Polymerization Enzymatic modification Bacterial synthesis FRET Turbidity 

References

  1. 1.
    Praefcke GJK (2017) Regulation of innate immune functions by guanylate-binding proteins. Int J Med Microbiol 308(1):237–245CrossRefGoogle Scholar
  2. 2.
    Praefcke GJK, McMahon HT (2004) The dynamin superfamily: universal membrane tabulation and fission molecules? Nat Rev Mol Cell Biol 5(2):133–147CrossRefGoogle Scholar
  3. 3.
    Praefcke GJK et al (1999) Nucleotide-binding characteristics of human guanylate-binding protein 1 (hGBP1) and identification of the third GTP-binding motif. J Mol Biol 292(2):321–332CrossRefGoogle Scholar
  4. 4.
    Ghosh A et al (2006) How guanylate-binding proteins achieve assembly-stimulated processive cleavage of GTP to GMP. Nature 440(7080):101–104CrossRefGoogle Scholar
  5. 5.
    Prakash B et al (2000) Structure of human guanylate-binding protein 1 representing a unique class of GTP-binding proteins. Nature 403(6769):567–571CrossRefGoogle Scholar
  6. 6.
    Kunzelmann S et al (2005) Nucleotide binding and self-stimulated GTPase activity of human guanylate-binding protein 1 (hGBP1). In: Balch WE, Der CJ, Hall A (eds) GTPases regulating membrane dynamics. Methods in enzymology, vol 404. Elsevier Academic Press, London, pp 512–527CrossRefGoogle Scholar
  7. 7.
    Schwemmle PSM (1994) The interferon-induced 67-kDa guanylate-binding protein (hGBP1) is a GTPase that converts GTP to GMP. J Biol Chem 268:11299–11305Google Scholar
  8. 8.
    Kunzelmann S et al (2006) Transient kinetic investigation of GTP hydrolysis catalyzed by interferon-gamma-induced hGBP1 (human guanylate binding protein 1). J Biol Chem 281(39):28627–28635CrossRefGoogle Scholar
  9. 9.
    Fres J et al (2010) Purification of the CaaX-modified, dynamin-related large GTPase hGBP1 by coexpression with farnesyltransferase. J Lipid Res 51(8):2454–2459CrossRefGoogle Scholar
  10. 10.
    Shydlovskyi S et al (2017) Nucleotide-dependent farnesyl switch orchestrates polymerization and membrane binding of human guanylate-binding protein 1. Proc Natl Acad Sci 114(28):5559–5568CrossRefGoogle Scholar
  11. 11.
    Cohen SN et al (1972) Nonchromosomal Antibiotic Resistance in Bacteria: Genetic Transformation of Escherichia coli by R-Factor DNA. Proc Natl Acad Sci 69(8):2110–2114CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Physical Chemistry I, Faculty of Chemistry and BiochemistryRuhr University BochumBochumGermany

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