Genetic Code Expansion with Unnatural Amino Acids: Biosynthesis and Incorporation of Halogen and Chalcogen-containing Amino Acids into Proteins

Abstract

Genetic code expansion (GCE) strategy allows incorporation of unnatural amino acids (UAAs) with diverse physical, chemical, and biochemical properties. Multiple UAAs have been successfully incorporated co-translationally with high fidelity and efficiency using a unique codon and a corresponding engineered aminoacyl-tRNA synthetase (aaRS)/tRNA pair. A critical feature of GCE is the orthogonality between the engineered pair and endogenous translational machinery of the host organism. This methodology serves as a powerful tool for studying protein structure and function both in vitro and in vivo, and for producing proteins with novel or enhanced properties. The first part of the thesis highlights the role of aaRS in catalyzing the attachment of amino acids (AAs) to their cognate tRNAs during protein synthesis. Due to the high specificity of their amino acid binding sites, each AA in the genetic code requires a distinct aaRS. In this study, we propose that aaRS can recognize halogenated UAAs via halogen bonding (XB). The stronger XB-forming ability of iodine, compared to chlorine or bromine, enables the selective incorporation of 3,5-diiodo-L-tyrosine into proteins. This work demonstrates that XB can be used to expand the genetic code and the aaRS can be engineered to discriminate structurally similar UAAs differing by a single atom. The second part of the thesis describes the advances in protein engineering enabled by genetic code expansion (GCE), while also addressing challenges associated with the chemical synthesis of unnatural amino acids (UAAs), such as complex synthesis, poor solubility, and reliance on organic solvents. To overcome these limitations, we have developed an in vivo strategy for the biosynthesis of tyrosine analogues. Specifically, we utilize Tyrosine Phenol Lyase (TPL) to produce chalcogen-containing tyrosine analogues, which are then incorporated into proteins using orthogonal translational machinery. This approach enables the simultaneous biosynthesis and incorporation of these analogues into proteins. The third part highlights the impact of chalcogen-containing tyrosine analogues on the fluorescence properties of green fluorescent protein (GFP), demonstrating how the introduction of a chalcogen atom into the chromophore can convert GFP into an orange fluorescent protein (OFP). We also investigated the redox behaviour of these GFP variants and their fluorescence response to oxidants and reductants. Notably, we successfully developed a circularly permuted GFP-66MeSeY variant that exhibits reversible fluorescence changes in response to oxidants such as H2O2 and reductants like GSH and L-cysteine. The last part of the thesis describes the role of the hydrogen-bonding network around the GFP chromophore in maintaining fluorescence. The introduction of MeSeY into the chromophore alters this network, yet fluorescence is retained. This disruption appears to be compensated by a chalcogen bond between the selenium atom of MeSeY and the oxygen of Ser205. We further demonstrate that mutating the residue at position 205 affects the spectral properties of GFP-66MeSeY, underscoring the importance of this interaction.