Protein synthesis with multiple non-canonical amino acids

生物细胞使用存储在基因和DNA中的遗传信息，从20种不同的氨基酸或构件中构建蛋白质。蛋白质在细胞中扮演着重要的结构和功能角色，例如，建立细胞之间的通信，将食物分子(如糖)转化为细胞可以利用的化学能，甚至复制或复制传递给下一代的遗传信息。从单细胞微生物到复杂的多细胞生物体，包括人类在内的各种生命都有类似的过程。**尽管蛋白质只由20种典型的氨基酸组成，但在成熟的蛋白质中发现了400多种不同的氨基酸。这些非典型氨基酸(NCAA)是生命所必需的，因为参与基本细胞过程的蛋白质经历了显著的翻译后修饰(PTM)。在这个过程中，某些(许多时候未知的)蛋白质被称为酶，在原始蛋白质形成后，对这20个组成单元的氨基酸进行化学修饰。PTMS传递改变细胞命运和基因表达的化学信号。PTM的确切作用或PTM的特定组合目前尚不清楚，因为还没有方法可用于合成具有多个NCAA的蛋白质。这种方法是迫切需要的，也是确定PTMS在蛋白质和途径中的结构和功能作用所必需的。**涉及PTM的一些最复杂和有趣的途径包括信号、表观遗传和氧化应激途径。在信号通路中，氨基酸与附着的磷酸盐以级联方式从一种蛋白质转移到另一种蛋白质(就像电话的分子游戏)。这个过程告诉细胞它的环境中发生了什么，能源在哪里，是否存在捕食者(如病毒)或猎物(在某些情况下是其他细胞)。表观遗传途径是另一个应用领域，涉及化学修饰的氨基酸，它改变了基因的表达，而实际上不改变基因组中的DNA序列。这些表观遗传变化实际上可以被下一代遗传。最后，依赖于NCAA硒半胱氨酸的氧化应激途径形成了细胞的天然抗氧化防御系统。由于硒半胱氨酸是一种化学活性很高的NCAA，拟议的工作还将探索使用这种NCAA来创建高效的生物催化剂(加速化学反应的试剂)。**为了研究这些蛋白质修饰在细胞途径中的作用并创造具有新特性的酶，拟议的研究将建立与多个NCAA合成蛋白质的系统。拟议的工作将涉及设计蛋白质合成系统，使蛋白质与多个NCAA定点硬连接，创建通往NCAA的新的生物合成和化学合成路线，以及设计工业相关应用的含硒半胱氨酸蛋白质。这项技术将是基础的，并不局限于这里概述的项目，因为NCAA，包括翻译后修饰，在自然界中无处不在，在从微生物到人类细胞的各种生物系统中发挥着重要作用。

The biological cell uses genetic information stored in genes and DNA to build proteins from 20 different amino acids or building blocks. Proteins play important structural and functional roles in the cell by, for example, establishing communication between cells, converting food molecules (like sugar) into chemical energy that the cell can use, and even copying or replicating the genetic information that is passed on to the next generation. Similar processes occur across the great diversity of life, from single-celled microbes to complex multicellular organisms, including humans.**Despite the fact that proteins are made from only 20 typical amino acids, over 400 different kinds of amino acids are found in mature proteins. These non-canonical amino acids (ncAAs) are essential to life because proteins involved in fundamental cellular processes undergo significant posttranslational modification (PTM). In this process, certain (many times unknown) proteins called enzymes chemically modify those 20 building block amino acids after the original protein is made. PTMs relay chemical signals that alter cell fate and gene expression. The precise role of PTMs or particular combinations of PTMs is currently unknown because no methods are available to synthesize proteins with multiple ncAAs. Such a method is urgently needed and essential to define the structural and functional role of PTMs in proteins and pathways. **Some of the most complex and interesting pathways involving PTMs include signalling, epigenetic and oxidative stress pathways. In signalling pathways, amino acids with attached phosphates are transferred in cascades from one protein to the next (like a molecular game of telephone). This process tells the cell what is going on in its environment, where energy sources are and whether predators (such as viruses) or prey (in some cases other cells) are present. Epigenetic pathways are another application area and involve chemically modified AAs that alter gene expression, without actually changing the sequence of DNA in the genome. These epigenetic changes can in fact be inherited by the next generation. Finally, oxidative stress pathways that rely on the ncAA selenocysteine form the cell's natural antioxidant defense system. Because selenocysteine is a highly chemically reactive ncAA, the proposed work will also explore using this ncAA to create efficient biocatalysts (agents that speed up chemical reactions). **To investigate the role of these protein modifications in cellular pathways and to create enzymes with novel properties, the proposed research will establish systems that synthesize proteins with multiple ncAAs. The proposed work will involve engineering protein synthesis systems to site-specifically hardwire proteins with multiple ncAAs, creating new biosynthetic and chemical synthetic routes to ncAAs, and designing selenocysteine containing proteins for industrially relevant applications. The technology will be fundamental and not limited to the projects outlined here, because ncAAs, including posttranslational modifications, are ubiquitous in nature and play important roles in diverse biological systems from microorganisms to human cells.