Near-natural Amino Acid Mutagenesis for the Engineering and Study of Protein Function

用于蛋白质功能工程和研究的近天然氨基酸诱变

• 批准号: 1603930
• 负责人: Alexander Deiters
• 金额: $ 45万
• 依托单位: University of Pittsburgh
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2020-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1603930&HistoricalAwards=false
• 关键词: Near; natural; Amino; Acid; Mutagenesis

1603930 Deiters, Alexander Most physiological processes rely on the function of proteins. In order to elucidate the precise biochemical mechanisms of protein function, researchers are increasingly turning to the tool set of unnatural amino acids that contain small structural or electronic changes which are very similar to Nature's set of common amino acids. These subtle differences, site-specifically incorporated into proteins, allow for the precise probing of protein structure and function. Within this project, new methodologies for the genetic encoding of these 'near-natural' amino acids will be developed and applied to the investigation of enzymatic mechanism. In order to elucidate the precise biochemical mechanisms of protein function, unnatural amino acids that contain small structural or electronic changes, e.g., in pKa, polarity, redox potential, H-bonding ability, nucleophilicity, isotope composition, etc, are versatile molecular probes. Although these 'near-natural' amino acids are extremely useful to investigate protein function when located near or in an active site, they are difficult to apply to the engineering of a biological system for the following reasons: 1) The chemical synthesis of proteins containing near-natural amino acids is laborious, expensive, and can routinely only be applied to small proteins. 2) The semi-synthesis of proteins can be technically challenging. 3) The use of auxotrophic bacterial or yeast strains in vivo leads to global, non-specific incorporation of near-natural amino acids into proteins preventing precise studies. 4) Near-natural amino acids often display toxicity due to global incorporation into the proteome. Within this project, a robust and general methodology for protein engineering through site-specific near-natural amino acid mutagenesis will be developed by temporarily disguising these amino acids as completely unnatural amino acids. This will be achieved by introducing a transient structural change into near-natural amino acids, thus 'hiding' them from the cellular machinery responsible for endogenous protein biosynthesis, but simultaneously providing a handle for the engineering of an orthogonal biosynthetic pathway. Specifically, introduction of protecting groups (caging groups) on near-natural amino acids converts them temporarily into completely unnatural amino acids, effectively solving the issue of non-specific incorporation into proteins in vivo. After site-specific incorporation into the protein of interest, the caging group is removed through an external trigger providing the original near-natural amino acid and thus near-natural protein. For the engineering of an orthogonal protein biosynthetic pathway, new tRNA synthetases for the in vivo incorporation of near-natural amino acids into proteins in pro- and eukaryotic cells are generated. The developed methodology will be applied to the precise investigation of enzyme active sites with atomic resolution.This award by the Biotechnology and Biochemical Engineering Program of the CBET Division is co-funded by the Division of Materials Research through BioMaPS funds.

1603930 Deiters，亚历山大大多数生理过程依赖于蛋白质的功能。为了阐明蛋白质功能的精确生化机制，研究人员越来越多地转向非天然氨基酸的工具集，这些非天然氨基酸包含与自然界常见氨基酸非常相似的小的结构或电子变化。这些细微的差异，位点特异性地结合到蛋白质中，允许精确探测蛋白质结构和功能。在这个项目中，将开发这些“近天然”氨基酸的遗传编码的新方法，并应用于酶机制的研究。为了阐明蛋白质功能的精确生化机制，含有小的结构或电子变化的非天然氨基酸，例如，在pKa、极性、氧化还原电位、氢键能力、亲核性、同位素组成等方面，是通用分子探针。尽管这些“近天然”氨基酸在位于活性位点附近或活性位点中时对于研究蛋白质功能极其有用，但由于以下原因，它们难以应用于生物系统的工程化：1）含有近天然氨基酸的蛋白质的化学合成是费力的、昂贵的，并且通常只能应用于小蛋白质。2)蛋白质的半合成在技术上具有挑战性。3)在体内使用营养缺陷型细菌或酵母菌株导致接近天然的氨基酸全面、非特异性地掺入蛋白质中，妨碍了精确的研究。4)近天然氨基酸通常由于整体并入蛋白质组而显示毒性。在该项目中，将通过将这些氨基酸暂时伪装成完全非天然的氨基酸来开发通过位点特异性近天然氨基酸诱变进行蛋白质工程的稳健和通用方法。这将通过将瞬时结构变化引入近天然氨基酸中来实现，从而将它们从负责内源性蛋白质生物合成的细胞机制中“隐藏”出来，但同时为正交生物合成途径的工程化提供了一个手柄。具体而言，在接近天然的氨基酸上引入保护基团（笼化基团）将其暂时转化为完全非天然的氨基酸，有效地解决了体内非特异性掺入蛋白质的问题。在位点特异性掺入到感兴趣的蛋白质中后，通过外部触发剂去除笼化基团，提供原始的近天然氨基酸，从而提供近天然蛋白质。对于正交蛋白质生物合成途径的工程化，产生了用于在原核细胞和真核细胞中将近天然氨基酸体内掺入蛋白质中的新tRNA合成酶。开发的方法学将应用于精确的原子分辨率的酶活性位点的调查。该奖项由CBET部门的生物技术和生物化学工程计划共同资助，由材料研究部门通过BioMaPS基金资助。