Stabilization of short collagen-like triple helices by protein engineering

Recombinant expression of collagens and fragments of collagens is often dif ficult, as their biosynthesis requires specific post-translational enzymes, in particular prolyl 4-hydroxylase. Although the use of hydroxyproline-def icient variants offers one possibility to overcome this difficulty, these p roteins usually differ markedly in stability when compared with the hydroxy proline-containing analogs. Here, we report a method to stabilize collagen- like peptides by fusing them to the N terminus of the bacteriophage T4 fibr itin foldon domain. The isolated foldon domain and the chimeric protein (Gl yProPro)(10)foldon were expressed in a soluble form in Escherichia coli. Th e recombinant proteins and the synthetic (ProProGly)(10) peptide were chara cterized by circular dichroism (CD) spectroscopy, differential scanning cal orimetry, and analytical ultracentrifugation. We show that the foldon domai n, 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 in dicated a cooperative all-or-none transition. As reported previously, (ProP roGly)(10) peptides form collagen triple helices of only moderate stability . When fused to the foldon domain, however, triple helix formation of (GlyP roPro)(10) is concentration independent, and the midpoint temperature of th e triple helix unfolding is significantly increased. The stabilizing functi on 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. (C) 2001 Academic Press.