Probing Protein Structure and Function with an Expanded Genetic Code

Abstract

A general biosynthetic method has been developed which makes it possible to site-specifically incorporate unnatural amino acids with novel properties into proteins. In this approach the codon encoding the amino acid of interest is replaced with the "blank" nonsense codon UAG by oligonucleotide-directed mutagenesis. A suppressor tRNA that recognizes this codon is generated by run-off transcription and then chemically aminoacylated with the desired unnatural amino acid. Addition of the mutagenized gene and the aminoacylated suppressor tRNA to an in vitro extract capable of supporting protein biosynthesis generates a mutant protein containing the unnatural amino acid at the specified position. This methodology has recently been used to study the stability, specificity, and catalytic properties of a number of proteins. In these studies amino acids and analogues possessing altered hydrogen-bonding, electronic. and steric properties and unique backbone conformations have all been site-specifically incorporated into proteins. In addition, photoactivatable amino acids, isotopically labeled amino acids, and amino acids bearing biophysical probes have been inserted site-specifically . This chemistry increases our ability to carry out detailed physical organic studies on this important class of macromolecules.

Synthetic biology projects in vitro

“…Nevertheless, toxicity, solubility, and purification issues encourage improvement of in vitro translation systems. The latter are also more versatile for incorporation of amino acid analogs: More than 100 have been incorporated at a single suppression site per protein (even carbohydrate linkages) (Cornish et al 1995;Zhang et al 2004). Crude E. coli systems with extended expression times have been enabled by continuous dialysis, but the efficiencies of translation of even homologous genes are unpredictable (Tian et al 2004).…”

Synthesis of Native Proteins by Chemical Ligation

“…Efforts have been made to use cell-free synthesis to expand the repertoire of ribosomal synthesis to include noncoded amino acids as building blocks (6,7). These attempts to incorporate other amino acids have had very limited successobtaining adequate amounts of pure protein from the cell-free translation systems can be a significant challenge (8), and many unnatural amino acids are simply not compatible with ribosomal polypeptide synthesis (9).…”

Site-specific, photochemical proteolysis applied to ion channels in vivo

“…A powerful tool for probing protein structure and function is the incorporation of unnatural amino acids by the method of nonsense suppression, a technique developed for in vitro translation systems by Schultz and coworkers (1)(2)(3). Recent work has expanded the scope of this methodology by establishing procedures for unnatural amino acid incorporation into proteins expressed in living cells (4)(5)(6)(7).…”