De novo design of artificial proteins with structural specificity as if they were native protains

为了设计具有天然蛋白质结构特异性的人工蛋白质，我们试图开发新的结构-序列相容功能和新的功能蛋白质设计方法，并用实验进行验证。此外，我们还讨论了点突变的稳定性分析和蛋白质折叠的模拟和实验。通过以上活动获得的洞察力被积累起来，以丰富设计策略。至于新的函数，我们开发了一个评估小段氨基酸在连续的Ramachandran图定义的局部结构上的适合性的函数。我们定义了12种类型的单部位构象编码(SSCC)，并推导了它们和氨基酸的统计势。使用这两种类型的诱饵集对该函数的精度进行了评估，我们注意到它的性能要好于使用DSSP的输出作为局部构象定义的函数。此外，我们还尝试了同源蛋白之间蛋白质功能的转换。基于肌红蛋白的3D结构，使用Moeller程序和3D-Profile对藻蓝蛋白序列进行了最小限度的修改。合成的序列含有部分α螺旋，每周结合一次HEM。另一种在藻蓝蛋白上挖洞用于折边的序列表现更好：具有更多的α螺旋含量和良好的折边结合。此外，我们还可以确定我们之前设计的人工Cro抑制物的核磁共振结构，并将其发表在《分子生物学杂志》上。

To design the artificial proteins that exhibit the structural specificity like naturally occurring proteins, we tried to develop the new structure-sequence compatibility functions and the new methodology to design functional proteins, and validate them with the experiments. Also we addressed to the stability analyses of point mutations and protein folding simulations and experiments. The insights obtained through these above activities are accumulated to enrich the design strategy. As for the new function, we developed one that evaluates the fitness of the small segment of amino acids on the local structure defined by the successive Ramachandran plot. We defined twelve types of Single Site Conformational Code (SSCC) and statistical potential was derived for them and amino acids. The accuracy of this function was estimated using the two types of decoy sets and we noticed its performance is better than the function that employs the output of DSSP as the definition of local conformation. Also we tried the transformation of the protein function between the homologous proteins. Based on the 3D structure of myoglobin, a phycocyanin sequence was minimally modified using the Modeller program and 3D-profile. The resulted sequence was synthesized and it has a part of α helices and binds HEM weekly. The sequence designed by the other method of digging a hole on phycocyanin for HEM, performs better : it has more α helical content and shows good HEM binding. Additionally, we could determined the NMR structure of artificial Cro repressor we designed before and published it in the Journal of Molecular Biology.