Graduate School of Science and Engineering, Yamagata University, 4-3-16 Jyonan, Yonezawa 992-8510, Yamagata, Japan.
Int J Mol Sci. 2022 Jan 9;23(2):701. doi: 10.3390/ijms23020701.
Top7 is a de novo designed protein whose amino acid sequence has no evolutional trace. Such a property makes Top7 a suitable scaffold for studying the pure nature of protein and protein engineering applications. To use Top7 as an engineering scaffold, we initially attempted structure determination and found that crystals of our construct, which lacked the terminal hexahistidine tag, showed weak diffraction in X-ray structure determination. Thus, we decided to introduce surface residue mutations to facilitate crystal structure determination. The resulting surface mutants, Top7sm1 and Top7sm2, crystallized easily and diffracted to the resolution around 1.7 Å. Despite the improved data, we could not finalize the structures due to high R values. Although we could not identify the origin of the high R values of the surface mutants, we found that all the structures shared common packing architecture with consecutive intermolecular β-sheet formation aligned in one direction. Thus, we mutated the intermolecular interface to disrupt the intermolecular β-sheet formation, expecting to form a new crystal packing. The resulting mutant, Top7sm2-I68R, formed new crystal packing interactions as intended and diffracted to the resolution of 1.4 Å. The surface mutations contributed to crystal packing and high resolution. We finalized the structure model with the R/R values of 0.20/0.24. Top7sm2-I68R can be a useful model protein due to its convenient structure determination.
Top7 是一种全新设计的蛋白质，其氨基酸序列没有进化痕迹。这种特性使 Top7 成为研究蛋白质本质和蛋白质工程应用的合适支架。为了将 Top7 用作工程支架，我们最初尝试确定结构，但发现我们构建的缺少末端六组氨酸标签的晶体在 X 射线结构测定中显示出较弱的衍射。因此，我们决定引入表面残基突变以促进晶体结构测定。由此产生的表面突变体 Top7sm1 和 Top7sm2 易于结晶，衍射分辨率约为 1.7 Å。尽管数据得到了改善，但由于高 R 值，我们仍无法最终确定结构。尽管我们无法确定表面突变体高 R 值的来源，但我们发现所有结构都具有共同的包装架构，连续的分子间β-折叠排列在一个方向上。因此，我们突变了分子间界面以破坏分子间β-折叠的形成，期望形成新的晶体堆积。预期的结果是，所得突变体 Top7sm2-I68R 形成了新的晶体堆积相互作用，并衍射至 1.4 Å 的分辨率。表面突变有助于晶体堆积和高分辨率。我们最终确定了结构模型，R 值和 R' 值分别为 0.20/0.24。由于其结构易于确定，Top7sm2-I68R 可以成为一种有用的模型蛋白。
