Design and laboratory evolution of artificial proteins

人工蛋白质的设计和实验室进化

• 批准号: 13650862
• 负责人: ISOGAI Yasuhiro
• 金额: $ 1.98万
• 依托单位: The Institute of Physical and Chemical Research
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13650862/
• 关键词: protein design; protein engineering; laboratory evolution; DNA binding proteins; heme proteins

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.

为了从一个新的角度阐明蛋白质结构的原理，这是传统的天然蛋白质生物化学所不能达到的，我们发展了一种设计具有天然性质的人工蛋白质的方法如下：1.分析了疏水氨基酸的侧链旋转异构体(旋转异构体)在蛋白质三级结构中的分布，推导出ΔS；，通过每个二级结构中残基-残基接触侧链的构象熵变化来寻找天然氨基酸序列结构独特性的来源，这可以作为设计具有独特结构的人工蛋白质的特异性参数。2.我们从蛋白质三维结构数据库中推导出一个新的蛋白质设计势能函数。这个功能可以评估每个氨基酸旋转异构体对蛋白质3D结构中的位置环境的适合性。利用这一功能，设计并合成了60个氨基酸组成的稳定主干三级结构的人工序列。人工合成的蛋白质折叠成目标二聚体，二级结构含量与天然λCro3相似。我们制备了生物素化的血红素，并研究了其在检测和纯化血红素蛋白中的应用。用生物素血红素从重组细胞提取物中纯化了天然和人工的血红素结合蛋白。因此，该分子对于设计的血红蛋白的实验室进化是有用的。

项目成果

Isogai, Y., Ota, M., Ishii, A., Ishida, M., Nishikawa, K.: "Identification of amino acids involved in protein structural uniqueness : Implication for de novo protein design"Protein Engineering. 15. 555-560 (2002)

Isogai, Y.、Ota, M.、Ishii, A.、Ishida, M.、Nishikawa, K.：“蛋白质结构独特性中涉及的氨基酸的鉴定：对从头蛋白质设计的启示”蛋白质工程。

Shiro, Y., Isogai, Y., Nakamura, H., Iizuka, T.: "Progress in Biotechnology, Vol.22(Endo, I. et al., Eds.),Physiological functions and molecular structures of new types of hemoproteins"Elsevier. 231(189-204) (2002)

Shiro, Y.、Isogai, Y.、Nakamura, H.、Iizuka, T.：“生物技术进展，第 22 卷（Endo, I. 等人编辑），新型生物的生理功能和分子结构

Isogai, Y.: "Stability and specificity of protein structure"RIKEN Review. 46. 48-49 (2002)

Isogai, Y.：“蛋白质结构的稳定性和特异性”RIKEN Review。

Shiro, Y., Isogai, Y., Nakamura, H., Iizuka, T.: "Physiological functions and molecular structures of new types of hemoproteins"Progress in Biotechnology. 22. 178-204 (2002)

Shiro, Y.、Isogai, Y.、Nakamura, H.、Iizuka, T.：“新型血红素蛋白的生理功能和分子结构”生物技术进展。

Shiro, Y., Isogai, Y., Nakamura, H., Iizuka, T.: "Physiological functions and molecular structures of new types of hemoproteins."Progress in Biotechnology. 22. 18-204 (2002)

Shiro, Y.、Isogai, Y.、Nakamura, H.、Iizuka, T.：“新型血红素蛋白的生理功能和分子结构。”生物技术进展。