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

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微小的结构或电子变化，与自然界的普通氨基酸非常相似。这些细微的差异，特异地结合到蛋白质中，使得对蛋白质结构和功能的精确探测成为可能。在这个项目中，将开发这些“接近天然”氨基酸的遗传编码的新方法，并将其应用于酶促机制的研究。为了阐明蛋白质功能的精确生化机制，含有微小结构或电子变化的非天然氨基酸，如pKa、极性、氧化还原电位、氢键能力、亲核性、同位素组成等，是多功能的分子探针。虽然这些“接近天然”的氨基酸在研究蛋白质功能时非常有用，当它们位于活性位点附近或处于活性位点时，它们很难应用于生物系统的工程，原因如下：1)含有接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。蛋白质的半合成在技术上具有挑战性。3)在体内使用营养不良细菌或酵母菌株会导致接近天然的氨基酸在蛋白质中全局性的、非特异性的结合，从而阻碍了精确的研究。4)接近天然的氨基酸通常由于整体结合到蛋白质组中而表现出毒性。在这个项目中，将通过暂时将这些氨基酸伪装成完全非天然的氨基酸，开发一种通过特定位点的近天然氨基酸诱变进行蛋白质工程的强大而通用的方法。这将通过在接近天然的氨基酸中引入瞬时结构变化来实现，从而将它们“隐藏”在负责内源性蛋白质生物合成的细胞机制之外，同时为正交生物合成途径的工程提供了一个处理方法。具体来说，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。在特定位点并入目标蛋白质后，笼化组通过提供原始的接近天然的氨基酸和接近天然的蛋白质的外部触发器被移除。为了设计一个正交的蛋白质生物合成途径，新的tRNA合成酶可以在原核和真核细胞中将接近天然的氨基酸整合到蛋白质中。所开发的方法将应用于酶活性位点的原子分辨率精确研究。该奖项由CBET部门的生物技术和生化工程项目颁发，由材料研究部通过BioMaPS基金共同资助。

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.