Unnatural Mutagenesis

In 1989, a biosynthetic in vitro method that allowed the site-specific incorporation of unnatural amino acids into proteins was introduced based on earlier work on nonsense suppression. The term "nonsense suppression" refers to the use of stop (nonsense) codons and suppressor transfer RNA (tRNA), which recognize stop codons. The method is based on the fact that only one of three stop codons in the genetic code is necessary for the termination of protein synthesis and the two unused stop codons can then be exploited for the introduction of unnatural amino acids.

The primary challenge in this technology is the generation of the modified suppressor tRNA with the unnatural amino acid (Figure 4.8). Once generated, the aa-tRNA is recognized by the mRNA carrying the specific stop codon, whereby the unnatural amino acid is incorporated into the protein at the specific position. Based on this principle, two slightly different methodologies have been developed for the site-specific incorporation of unnatural amino acids: one method applies tRNAs that are chemically aminoacylated with the unnatural amino acid of interest, and the aa-tRNA is subsequently applied in an expression system to generate the protein of interest (Figure 4.8). The other method employs the development of pairs of orthogonal tRNA and aminoacyl-tRNA synthetases (aaRS), where the latter is developed so that it selectively recognizes aminoacylate an unnatural amino acid.

In the chemical aminoacylation of tRNA, a dinucleotide is prepared by chemical synthesis and subsequently aminoacylated with the unnatural amino acid of interest. The aa-tRNA is obtained by the ligation of a truncated tRNA where a dinucleotide at the 3'-terminus is missing with the prepared aminoacylated dinucleotide (Figure 4.8). If an in vitro expression system is used, the aa-tRNA is simply added to the media, and when whole cell expression systems are used, the aa-tRNA is injected into the cell. A particular attractive expression system for this methodology is Xenopus oocytes, which is generally used for electrophysiological studies of ion channels, receptors, and transporters. The oocyte is coinjected with two RNA species: the modified mRNA encoding for the target protein and the aa-tRNA chemically acylated with an unnatural amino acid. This coinjection results in synthesis and surface expression of the target protein containing the unnatural amino acid (Figure 4.8).

The methodology has been used to incorporate a large number of structurally diverse unnatural amino acids, representing a large variety of functionalities, into proteins. In most cases the unnatural amino acids have been a-amino acids but also non-a-amino acids and most notably a-hydroxy acids have been incorporated with the latter introducing an amide-to-ester mutation in the protein backbone (Figure 4.9). These studies have shown that translation factors and the ribosome are compatible with many types of unnatural amino acids.

In studies of ligand-gated ion channels, such as nicotinic acetylcholine (nACh), g-aminobutyric acid (GABA), and serotonin (5-HT3) receptors (see Chapters 12 and 14), the technology has proven particularly valuable. These studies were pioneered by Dougherty and Lester, who have explored the molecular details of the cation-p interaction between the quaternary ammonium group of acetylcholine and aromatic residues in the nACh receptor; this was achieved by the site-specific incorporation of fluoro-substituted tyrosine and tryptophan residues, where the fluoro substituent gradually decreases the ability of the aromatic moiety to interact in cation-p interactions. These studies have provided unique details of acetylcholine interaction with subtypes of nACh receptors at the molecular level.

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