Study Of The Carbamate&Formyl Lysine PTM Through Genetically Encoded Unnatural Amino Acid

Over the recent ten years, the unnatural amino acid (UAA) technology based on genetic code expansion has been widely applied. Through this technology, we can incorporate the target UAA into the specific site of protein of interest. The technology was widely used from prokaryote (bacteria) to eukaryote (Saccharomyces, mammalian cell and Arabidopsis thaliana). Over100UAAs were genetically expanded in different systems. Various purposes were realized making use of the property of UAAs themselves and the interaction between UAA and proteins. For example, the protein was labeled site specifically with different kinds of biological or chemical probes to study the nuclear magnetic resonance, infrared spectroscopy, FRET, fluorescence, etc. The enzyme activity was improved by incorporating UAA. Post translational modification (PTM) was introduced into the specific site of protein by UAA to study the function. The incorporation of UAA into protein was dependent on the recognition of the Pyl aaRS binding pocket with the UAA. The critical residues of the binding pocket was mutated to construct a library of PyIRS. The positive and negative screening strategy were applied to search the proper aaRS which can recognize the target UAA. The UAA was incorporated into the specific site of the protein to study the change of function and structure.Most of the living organisms on earth were relied on the biological carbon dioxide fixation. Rubisco (Ribulose-1,5-bisphosphate carboxylase/oxygenase), the first enzyme in the Calvin cycle of CO2fixation, performs the catalysis with a rather low rate, which is a rate-limiting step in the overall carbon fixation. There is an important PTM named carbamate lysine (Kcx) which can fix and activate Mg2+. For the first time we studied the functional role of the PTM by UAA technology. We hope that the enzyme activity of rubisco could be improved to enhance the photo synthesis efficiency, with the ultimate goal of increasing the crop yield. We begin with designing a series of structural analogues of Kcx. The phenol ester and photo caged group were introduced to protect the terminal carboxyl group. After screening, the PylRS mutant that can recognize the UAA was identified. The UAA was successfully incorporated into the189TAG of rubsico to simulate the Kcx PTM. The result indicates that the enzyme activity of rubisco harboring the UAA in189site was almost gone. We can conclude that the Kcx PTM is indispensable for the catalysis function of rubisco. The amino group in the side chain of Kcx plays a more important role than thought before.Lysine formylation is a newly-discovered posttranslational modification in histones and other nuclear proteins, which has a well-recognized but poorly defined role in chromatin conformation modulation and gene expression. To date, there is no general method to site-specifically incorporate NE-formyllysine into a defined site of proteins. Through the artifacial evolution of PylRS, for the first time we report the highly efficient genetic incorporation of the unnatural amino acid Nε-formyllysine into proteins produced in Escherichia coli and mammalian cells by using an orthogonal Nε-formyllysine tRNAsynthetase/tRNAcuA pair. The recognization for acetyllysine and formyllysine by antibody is studied. The data shows that the anti-acetyllysine antibody does not recognize protein bearing formyllysine, which indicates that formyllysine posttranslational modification may play a different role from acetyllysine. This technique can be further applied to study the role of lysine formylation in epigenetic regulation as a powerful tool.