title: In vivo incorporation of non-canonical amino acids by using the chemical aminoacylation strategy: a broadly applicable mechanistic tool.
abstract: We describe a strategy for incorporating non-canonical 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 monitoring protein function, primarily by electrophysiology. With this protocol, a very wide range of non-canonical amino acids can be employed, allowing both systematic structure-function studies and the incorporation of reactive functionalities. 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.
authors.affiliation: Division of Chemistry and Chemical Engineering, California Institute of Technology, 1200 E. California Blvd., Pasadena, CA 91125 (USA). dadougherty@caltech.edu.
authors.forename: Dennis A
authors.initials: DA
authors.lastname: Dougherty
authors.forename: Ethan B
authors.initials: EB
authors.lastname: Van Arnam
mesh_terms: D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism*; D000818:Animals; D006801:Humans; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry* / Q000378:metabolism; D046249:Transfer RNA Aminoacylation
chemical_list: D000596:Amino Acids; D011506:Proteins; D012346:RNA, Transfer, Amino Acyl
keywords: Xenopus oocytes; backbone mutations; cation-pi interactions; fluorescence; nonsense suppression