Protein synthesis with multiple non-canonical amino acids

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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.