Stabilization of short collagen-like triple helices by protein engineering

Sabine Frank, Richard A. Kammerer, Diane Mechling, Therese Schulthess, Ruth Landwehr, James Bann, Yuan Guo, Ariel Lustig, Hans Peter Bächinger, Jürgen Engel

    Research output: Contribution to journalArticlepeer-review

    Abstract

    Recombinant expression of collagens and fragments of collagens is often difficult, as their biosynthesis requires specific post-translational enzymes, in particular prolyl 4-hydroxylase. Although the use of hydroxyproline-deficient variants offers one possibility to overcome this difficulty, these proteins usually differ markedly in stability when compared with the hydroxyproline-containing analogs. Here, we report a method to stabilize collagen-like peptides by fusing them to the N terminus of the bacteriophage T4 fibritin foldon domain. The isolated foldon domain and the chimeric protein (GlyProPro)10foldon were expressed in a soluble form in Escherichia coli. The recombinant proteins and the synthetic (ProProGly)10 peptide were characterized by circular dichroism (CD) spectroscopy, differential scanning calorimetry, and analytical ultracentrifugation. We show that the foldon domain, which comprises only 27 amino acid residues, forms an obligatory trimer with a high degree of thermal stability. The CD thermal unfolding profiles recorded from foldon are monophasic and completely reversible upon cooling. Similar Van't Hoff an calorimertic enthalpy values of trimer formation indicated a cooperative all-or-none transition. As reported previously, (ProProGly)10 peptides form collagen triple helices of only moderate stability. When fused to the foldon domain, however, triple helix formation of (GlyProPro)10 is concentration independent, and the midpoint temperature of the triple helix unfolding is significantly increased. The stabilizing function of the trimeric foldon domain is explained by the close vicinity of its N termini, which induce a high local concentration in the range of 1 M for the C termini of the collagen-like-peptide. Collagen-foldon fusion proteins should be potentially useful to study receptor-collagen interactions. © 2001 Academic Press.
    Original languageEnglish
    Pages (from-to)1081-1089
    Number of pages8
    JournalJournal of molecular biology
    Volume308
    Issue number5
    DOIs
    Publication statusPublished - 18 May 2001

    Keywords

    • Collagen-like peptides
    • Foldon
    • Oligomerization domain
    • Protein engineering
    • Stabilization

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