Genetic incorporation of unnatural amino acids for the synthesis of proteins with defined modifications

Residue-specific incorporation and site-specific incorporation methods are widely used to incorporate an increasing number of novel unnatural amino acids (UAAs) into proteins with great potential to facilitate their structure-function studies. This dissertation presents my work on the genetic incorporation of several UAAs by using these two methods into proteins and their applications in the synthesis of proteins with defined modifications, which are important for understanding their role in cellular events. These two methods together with other protein synthesis and modification techniques are introduced in Chapter 1 of this thesis. In chapter 2, a residue-specific incorporation method was used to incorporate azidonorleucine (ANL) and azidonorvaline (ANV) into proteins via methionine auxotrophic strain. The genetically incorporated ANL served as the orthogonal lysine precursor in the protein, onto which one ligatable auxiliary group was installed for native chemical ubiquitination. The semisynthesis of diubiquitin and its characterization was demonstrated. To further expand the utility of our method, ubiquitylated H2A was also synthesized efficiently in a similar way, which was used to study the behavior of several deubiquitinases towards it and the crosstalk between H2A ubiquitination and H3K36 methylation. In this chapter, we also attempted to develop one new method to prepare arginine methylated protein using genetically incorporated ANV. One mutant MetRS that can efficiently activate ANV was found. Several guanidinylating reagents were synthesized and tested for the site-specific guanidinylate ion at the protein level. The pyrrolysyl-tRNA synthetase (PylRS)/PylT pair has been wildly used to incorporate site-specifically UAAs into proteins in E. coli. In chapter 3, several pyrrolysine analogs were shown to be incorporated into proteins via orthogonal PylRS/PylT pair. One genetically incorporated α-hydroxyl amino acid was used to synthesize protein α-thioester by utilization of cysteinyl prolyl ester (CPE) auto-activating motif on protein. The proposed formation of diketopiperazine thioester via an intramolecular N–S acyl shift reaction was not successful possibly due to the steric hindrance of the α-hydroxyl amino acid located in our CPE motif. One genetically incorporated Cbz-protected homocysteine was used to synthesize histones with two different modifications by orthogonal cysteine-based chemistry. One model H3 protein was shown to be installed efficiently with a dimethylated lysine mimic at K27. The attempt to install the second modification into H3 failed due to the poor efficiency of the deprotection of Cbz-protected homocysteine using silver acetate or iodine in the acetic acid buffer. In the last part of this chapter, to incorporate more new UAAs into protein using PylRS/tRNA pair, the two plasmids based selection system was established in our lab. The construction and functional test of two plasmids used in the positive and negative selections were presented. Two libraries of mutant PylRS was constructed and successfully used to screen the mutant that can recognize the pyrrolysine analogs.​Doctor of Philosophy (SBS

Native chemical ubiquitination using a genetically incorporated azidonorleucine

A robust chemical ubiquitination method was developed. The method employed a genetically incorporated azidonorleucine as an orthogonal lysine precursor for the installation of a Gly residue bearing an Nα-auxiliary which mediated the ligation between ubiquitin(1–75)-thioester and the target protein. To demonstrate our methodology, a model protein, K48-linked diubiquitin, was synthesized with an overall yield of 35%.ASTAR (Agency for Sci., Tech. and Research, S’pore)Accepted versio

Immobilization and intracellular delivery of circular proteins by modifying a genetically incorporated unnatural amino acid

Backbone-cyclic proteins are of great scientific and therapeutic interest owing to their higher stability over their linear counterparts. Modification of such cyclic proteins at a selected site would further enhance their versatility. Here we report a chemoenzymatic strategy to engineer site-selectively modified cyclic proteins by combining butelase-mediated macrocyclization with the genetic code expansion methodology. Using this strategy, we prepared a cyclic protein which was modified with biotin or a cell-penetrating peptide at a genetically incorporated noncanonical amino acid, making the cyclization-stabilized protein further amenable for site-specific immobilization and intracellular delivery. Our results point to a new avenue to engineering novel cyclic proteins with improved physicochemical and pharmacological properties for potential applications in biotechnology and medicine

Thiazolidine-Masked α‑Oxo Aldehyde Functionality for Peptide and Protein Modification

α-Oxo aldehyde-based bioconjugation chemistry has been widely explored in peptide and protein modifications for various applications in biomedical research during the past decades. The generation of α-oxo aldehyde via sodium periodate oxidation is usually limited to the <i>N</i>-terminus of a target protein. Internal-site functionalization of proteins with the α-oxo aldehyde handle has not been achieved yet. Herein we report a novel method for site-specific peptide and protein modification using synthetically or genetically incorporated thiazolidine-protected α-oxo aldehyde. Efficient unmasking of the aldehyde was achieved by silver ion-mediated hydrolysis of thiazolidine under mild conditions for the first time. A model peptide and a recombinant protein were used to demonstrate the utility of this new method, which were site-specifically modified by oxime ligation with an oxyamine-functionalized peptide labeling reagent. Therefore, our current method has enriched the α-oxo aldehyde synthetic tool box in peptide and protein bioconjugation chemistry and holds great potential to be explored in novel applications in the future

Cu(II)/THPTA-Mediated Thiazolidine Deprotection for Living Phages and Cell Surfaces Labeling

Incorporating unnatural bioorthogonal groups into peptides and proteins offers an excellent opportunity to endow them with new properties in a precise and controlled manner. Among these, the α-oxo aldehyde group is particularly suitable for the post-functionalization of peptides and proteins due to its versatility and stability in aqueous buffers. However, the facile and site-specific incorporation of α-oxo aldehyde into proteins, especially in living systems, remains a long-lasting challenge. Here, we describe a novel Cu(II)/THPTA-Mediated Thiazolidine Deprotection (CUT-METHOD) strategy for post-installation of a highly-active α-oxo aldehyde moiety, which is released from a thiazolidine ring borne by a genetically encoded unnatural amino acid ThzK. This reaction is performed under physiological conditions, thereby enabling the chemoselective and site-specific modification of proteins via oxime ligation without compromising their integrity and function. To validate its versatility, we successfully performed site-specific incorporation of α-oxo aldehyde into recombinant proteins and those displayed on M13 filamentous bacteriophage particles and bacterial cell surfaces. In addition, by leveraging Spycatcher/Spytag chemistry and oxime ligation, the bacterial cells bearing aldehyde generated via the CUT-METHOD could be simultaneously decorated with two distinct functional molecules, providing a novel one-pot dual labeling platform for the construction of living bacterial cell-based cancer targeting systems. Put together, we have demonstrated that the CUT-METHOD strategy is a significant addition to the current bioorthogonal chemistry toolbox with broad applications anticipated in the near future

Thiazolidin-5-imine Formation as a Catalyst-Free Bioorthogonal Reaction for Protein and Live Cell Labeling

A previously undescribed reaction involving the formation of a thiazolidin-5-imine linkage was developed for bio-conjugation. Being highly specific and operating in aqueous media, this simple condensation reaction is used to chemoselectively label peptides, proteins and living cells under physiological conditions without the need to use toxic catalysts or reducing reagents

Tagging Transferrin Receptor with a Disulfide FRET Probe To Gauge the Redox State in Endosomal Compartments

Although the basic process of receptor-mediated endocytosis (RME) is well established, certain specific aspects, like the endosomal redox state, remain less characterized. Previous studies used chemically labeled ligands or antibodies with a FRET (fluorescence resonance energy transfer) probe to gauge the redox activity of the endocytic pathway with a limitation being their inability to track the apo receptor. New tools that allow direct labeling of a cell surface receptor with synthetic probes would aid in the study of its endocytic pathway and function. Herein, we use a peptide ligase, butelase 1, to label the human transferrin receptor 1 (TfR1) in established human cell lines with a designer disulfide FRET probe. This strategy enables us to obtain real-time live cell imaging of redox states in TfR1-mediated endocytosis, attesting a reducing environment in the endosomal compartments and the dynamics of TfR1 trafficking. A better understanding of endocytosis of different cell surface receptors has implications in designing strategies that hijack this natural process for intracellular drug delivery.Ministry of Education (MOE)Accepted versionThis research is supported by the Ministry of Education (MOE 2016-T3-1-003) of Singapore and by the Singapore National Research Foundation under its Antimicrobial Resistance IRG administered by the Singapore-MIT Alliance for Research and Technology