EXPANDING PROTEIN SEQUENCE SPACE THROUGH INCORPORATION OF NON-CANONICAL AMINO ACIDS

Abstract

Protein sequence space has been augmented by researchers wanting to expand the diversity of chemical functionalities that can exist within proteins. Artificial amino acids can range from simple atom substitutions such as fluorination, to installation of reactive handles like azides and alkynes. Researchers build upon the framework of natural proteins and have developed methods of installing a wide variety of artificial amino acids into proteins. Chapter 1 discusses in detail the two methods for metabolic incorporation of unnatural amino acids: site-specific incorporation and residue-specific incorporation. Advantages and disadvantages to each method are detailed as well as applications of these methods to the examination of problems in chemical biology. Non-canonical amino acids analogues that are structurally similar to their canonical counterparts can be recognized by the endogenous translational machinery for residue-specific incorporation. Chapter 2 describes the directed evolution of the methionyl-tRNA synthetase (MetRS) to incorporate propargylglycine, an alkyne analogue that is not recognized by the wild-type MetRS. A new MetRS variant active towards propargylglycine was identified after screening libraries of both active site mutations and error-prone PCR mutations. PraRS is capable of producing proteins where methionine is quantitatively replaced by propargylglycine. PraRS also does not recognize azidonorleucine, an azide methionine analogue for which the NLL-MetRS was evolved in order to enable cell-specific protein labeling. A method to identify cellular origins of proteins from two different bacterial strains in co-culture was developed using the NLL-MetRS and PraRS. hapter 3 illustrates the effects of global incorporation of non-canonical amino acids into globular proteins. Although trifluoroleucine and homoisoleucine have shown to increase the thermostability of model proteins, incorporation into more chloramphenicol acetyltransferase (CAT) does not yield the same benefits. We find that mutations that stabilize CAT for fluorinated amino acid incorporation do not protect against homoisoleucine incorporation. Lastly, access to new chemical reactions for protein modification requires synthesis and incorporation of new non-canonical amino acid analogues. Chapter 4 describes the design of two new artificial amino acids, S-allyl-homocysteine and 3-furanylalanine, for residue specific incorporation without expression of mutant synthetases. Also, a third amino acid, azidomethylphenylalanine, was designed for activation by a previously discovered phenylalanine-tRNA synthetase mutant. Incorporation of these three analogues provides chemical handles that are potential reagents for cross metathesis, Diels-Alder cycloaddition, and generation of a molecular epitope for binding to synthetic receptors.