GIGA-Research, Molecular Biology and Genetic Engineering Unit, University of Liège, Liège, Belgium.
PLoS One. 2013 Aug 19;8(8):e71858. doi: 10.1371/journal.pone.0071858. eCollection 2013.
The computational protein design protocol Rosetta has been applied successfully to a wide variety of protein engineering problems. Here the aim was to test its ability to design de novo a protein adopting the TIM-barrel fold, whose formation requires about twice as many residues as in the largest proteins successfully designed de novo to date. The designed protein, Octarellin VI, contains 216 residues. Its amino acid composition is similar to that of natural TIM-barrel proteins. When produced and purified, it showed a far-UV circular dichroism spectrum characteristic of folded proteins, with α-helical and β-sheet secondary structure. Its stable tertiary structure was confirmed by both tryptophan fluorescence and circular dichroism in the near UV. It proved heat stable up to 70°C. Dynamic light scattering experiments revealed a unique population of particles averaging 4 nm in diameter, in good agreement with our model. Although these data suggest the successful creation of an artificial α/β protein of more than 200 amino acids, Octarellin VI shows an apparent noncooperative chemical unfolding and low solubility.
计算蛋白质设计方案 Rosetta 已成功应用于各种蛋白质工程问题。在这里，我们的目的是测试它从头设计采用 TIM 桶折叠的蛋白质的能力，这种折叠需要的残基数大约是迄今为止成功从头设计的最大蛋白质的两倍。设计的蛋白质 Octarellin VI 包含 216 个残基。它的氨基酸组成与天然 TIM 桶蛋白相似。当生产和纯化时，它表现出远紫外圆二色性光谱，具有折叠蛋白的特征，具有α-螺旋和β-折叠二级结构。其稳定的三级结构通过色氨酸荧光和近紫外圆二色性得到证实。它在 70°C 下表现出热稳定性。动态光散射实验显示出一种独特的粒子群体，平均直径为 4nm，与我们的模型非常吻合。尽管这些数据表明成功地创造了一种超过 200 个氨基酸的人工α/β 蛋白，但 Octarellin VI 表现出明显的非协同化学展开和低溶解度。
