In Vivo Incorporation of Unnatural Amino Acids Using the Chemical Aminoacylation Strategy. A Broadly Applicable Mechanistic Tool

We describe one strategy for incorporating unnatural amino acids site-specifically into proteins expressed in living cells, involving organic synthesis to chemically aminoacylate a suppressor tRNA, protein expression in Xenopus oocytes, and, primarily, monitoring protein function by electrophysiology. With this protocol, a very wide range of unnatural amino acids can be employed, allowing both systematic structure-function studies and the incorporation of reactive functionality. Here we present an overview of the methodology and examples meant to illustrate the versatility and power of the method as a tool for investigating protein structure and function.