Synthesis of Proteins with Homogenous Chemical and Posttranslational Modifications
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Genetic encoding non-canonical amino acids (NCAAs) is a facile approach to synthesize proteins with homogenous modifications. In my graduate study, I demonstrated the application of this approach in the synthesis of a variety of proteins with site-specific chemical and post-translational modifications. My first effort was to synthesize and demonstrate the genetic installation of N^(ԑ)-methyl-L-lysine (Kme1) into proteins indirectly via the incorporation of N^(ԑ)-(2-Nitrobenzyl)oxycarbonyl-N^(ԑ)-methyl-L-lysine and its following photolytic deprotection to recover Kme1. This novel method has been applied in the Liu group at Texas A&M Department of Chemistry for understanding epigenetics roles of nucleosomal post-translational lysine modifications. In order to develop an approach for easy access to proteins with defined two-color labeling, the next endeavor in my graduate study was to design and demonstrate a novel method for the genetic encoding of two different NCAAs in one protein that can be further modified to introduce two dyes with different colors. I showed that NCAAs with two different functionalities could be successfully incorporated into one protein using a model protein glutamine binding protein (QBP), I demonstrated two dyes that form a Förster resonance energy transfer (FRET) pair could be successfully covalently linked to two NCAAs in a one-pot and catalyst-free fashion. The synthesized two-dye labeled QBP was successfully applied to undergo the folding/unfolding analysis of QBP in the presence of guanidinium. I was also involved in the synthesis of proteins with hydroxylamine and hydrazine groups for their efficient labeling with ketos. I synthesized Nԑ-allyl protected hydroxylamine- and hydrazine-containing NCAAs and devised a method for their incorporation into superfolder green fluorescent protein (sfGFP). Similarly to the genetic encoding of Kme1, the direct incorporation of N^(ԑ), N^(ԑ)-dimethyl-L-lysine (Kme2) also suffers from its structural similarity with lysine. In order to genetically install Kme2 in proteins, we aimed to genetically incorporate allysine into proteins followed by reductive amination with dimethylamine to make Kme2. Several allysine precursors were synthesized. I demonstrated that they could be site-selectively incorporated into proteins and deprotected to recover allysine. The Liu group is now in the process to carry out the reductive amination reaction to synthesize proteins with Kme2. An important biological study I was involved in was to synthesize histone H3 with different acylation types at its multiple lysines and then I used these proteins to probe substrate and modification type specificities of one group of histone deacetylases, sirtuins. H3 variants with four modification types, acetylation, propionylation, butyrylation, and crotonylation at about 10 lysine sites were recombinantly expressed. They were used to probe sirtuin enzymes. Based on our results, we conclude that Sirt1 and Sirt2 act as universal histone deacylases regardless of lysine sites or modification types. In contrast Sirt6 and Sirt7 barely show any reactivity toward any modification types or lysine sites we tested.
Wu, Bo (2014). Synthesis of Proteins with Homogenous Chemical and Posttranslational Modifications. Doctoral dissertation, Texas A & M University. Available electronically from