Design and laboratory evolution of artificial proteins

为了从一个新的角度揭示蛋白质结构的原理，这是天然蛋白质的正统生物化学无法达到的，我们开发了一种方法来设计具有天然类似性质的人造蛋白质，如下所示。通过分析疏水氨基酸侧链旋转异构体（rotamers）在蛋白质三级结构中的分布，推导出各二级结构中残基-残基接触对侧链构象熵的变化ΔS^<contact>，以寻找天然氨基酸序列结构独特性的来源，可作为设计具有独特结构的人工蛋白质的特异性参数。我们从蛋白质三维结构数据库中导出了一个新的蛋白质设计势函数。该功能可以评估蛋白质三维结构中每个氨基酸旋转体对位点环境的适应性。利用该函数，设计并合成了60个稳定主链三级结构的人工氨基酸序列。合成的人工蛋白折叠成目标二聚体，二级结构含量与天然的λ cr3相似。我们制备了生物素化血红素，并对其用于血红蛋白的检测和纯化进行了研究。用生物素血红素从重组细胞提取物中纯化天然和人工血红素结合蛋白。因此，该分子可用于设计血红蛋白的实验室进化。

To shed light on the principles of protein architecture in a new aspect that is unattainable by orthodox biochemistry of natural proteins, we have developed a methodology to design artificial proteins with native-like properties as follows.1.We analyzed the distribution of the side-chain rotational isomers (rotamers) of hydrophobic amino acids in protein tertiary structures and derived ΔS^<contact>, the conformational-entropy changes of side chains by residue-residue contacts in each secondary structure to find the origin of the structural uniqueness in native amino acid sequences, which can be used as a specificity parameter for designing artificial proteins with a unique structure.2.We derived a new potential function for protein design from a protein 3D structural database. This function can assess fitness of each amino-acid rotamer to a site environment in protein 3D structures. By using this function, an artificial sequence of the sixty amino acids to stabilize the backbone tertiary structure was designed and synthesized. The synthesized artificial protein folds into the targeted dimer with the secondary structure contents similar to those of native λCro.3.We prepared biotinylated heme and investigated its usage for detection and purification of hemoproteins. The purification of native and artificial heme-binding proteins from recombinant cell extracts using biotinyl heme was demonstrated. Thus the molecule is useful for laboratory evolution of designed hemoproteins.