Stabilization of short collagen-like triple helices by protein engineering
Stabilization of short collagen-like triple helices by protein engineering
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DOI：
10.1006/jmbi.2001.4644
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发表时间：
2001-05-18
影响因子：
5.6
通讯作者：
Engel, J
中科院分区：
文献类型：
--
作者：
Frank, S;Kammerer, RA;Engel, J
胶原蛋白和胶原蛋白片段的重组表达通常是困难的，因为它们的生物合成需要特定的翻译后酶，特别是Pro-4-羟基酶。虽然使用缺乏羟脯氨酸的变异体提供了一种克服这一困难的可能性，但与含有羟脯氨酸的类似物相比，这些蛋白质在稳定性上通常有明显的不同。在这里，我们报道了一种通过将胶原样肽融合到噬菌体T4纤维蛋白折叠结构域的N端来稳定胶原蛋白样肽的方法。分离的Foldon结构域和嵌合蛋白(GlyProPro)(10)Foldon在大肠杆菌中以可溶性形式表达。用圆二色谱(CD)、差示扫描量热法和分析超速离心法对重组蛋白和合成的(ProProGly)(10)肽进行了表征。我们发现，只有27个氨基酸残基的Foldon结构域形成了一个具有高度热稳定性的必需三聚体。从褶皱带记录的CD热展开剖面是单相的，冷却时完全可逆。相似的Van‘t Hoff和卡马尔三聚体生成热值表明了一个要么全有要么全无的协同转变。正如先前报道的那样，(ProProGly)(10)肽形成的胶原蛋白三螺旋只有中等稳定性。然而，当融合到折叠结构域时，(GlyProPro)(10)的三螺旋形成与浓度无关，并且三螺旋展开的中点温度显著提高。三聚体Foldon结构域的稳定作用是由于它的N末端附近，导致胶原样肽的C末端在1M范围内有很高的局部浓度。胶原-折叠融合蛋白在研究受体-胶原相互作用方面具有潜在的应用价值。(C)2001年学术出版社。
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. (C) 2001 Academic Press.