Stabilization of short collagen-like triple helices by protein engineering
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)(10)foldon 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 and 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.
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- Authors
- Frank, S., Kammerer, R. A., Mechling, D., Schulthess, T., Landwehr, R., Bann, J., Guo, Y., Lustig, A., Bächinger, H. P., & Engel, J.
- Affiliations
- University of Basel (CH)
- Shriners Hospitals for Children - Portland (US)
- Washington University in St. Louis (US)
- Corresponding author
- Engel, Jürgen
- Venue
- Journal of Molecular Biology
- Publisher
- Elsevier BV
- Published
- 2001-05
- Volume
- 308
- Issue
- 5
- Pages
- 1081-1089
- ISSN
- 0022-2836 (ISSN-L)
- OpenAlex
- W2037490596
- Cited by
- 213
- References
- 20
- Citations by year
- 2026: 8 · 2025: 11 · 2024: 2 · 2023: 8 · 2022: 5 · 2021: 6 · 2020: 5 · 2019: 12 · 2018: 2 · 2017: 7 · 2016: 3 · 2015: 14 · 2014: 5 · 2013: 16 · 2012: 12
- FWCI
- 8.51
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Journal of Molecular Biology (publishedVersion)
- PubMed (publishedVersion)
- Research Explorer (The University of Manchester) (submittedVersion)
- Funders
- Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung
- Topics
- Physical Sciences › Materials Science › Biomaterials › Collagen: Extraction and Characterization
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
- Keywords
- protein stabilization, collagen-like peptides, protein trimerization, thermal stability, prolyl 4-hydroxylase, chimeric fusion protein
- MeSH terms
- Amino Acid Sequence, Biopolymers, Calorimetry, Differential Scanning, Circular Dichroism, Collagen, Escherichia coli, Molecular Sequence Data, Peptides, Recombinant Fusion Proteins, Solubility, Temperature, Thermodynamics, Ultracentrifugation, Viral Proteins, Protein Engineering, Bacteriophage T4, Protein Structure, Secondary, Protein Structure, Tertiary, Protein Structure, Quaternary
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