A General Method for Site-specific Incorporation of Unnatural Amino Acids into Proteins

Abstract

A new method has been developed that makes it possible to site-specifically incorporate unnatural amino acids into proteins. Synthetic amino acids were incorporated into the enzyme beta-lactamase by the use of a chemically acylated suppressor transfer RNA that inserted the amino acid in response to a stop codon substituted for the codon encoding residue of interest. Peptide mapping localized the inserted amino acid to a single peptide, and enough enzyme could be generated for purification to homogeneity. The catalytic properties of several mutants at the conserved Phe66 were characterized. The ability to selectively replace amino acids in a protein with a wide variety of structural and electronic variants should provide a more detailed understanding of protein structure and function.

Gelatinase-mediated migration and invasion of cancer cells

“…The two main strategies are the residue-specific incorporation of nonnatural amino acids by misaminoacylation of transferRNAs (tRNAs) (Kiick et al, 2001;Kirshenbaum et al, 2002;Niemz and Tirrell, 2001) and the site-directed incorporation, which utilizes non-cognate amber suppressor tRNA/aminoacyl-tRNA synthetase pairs (Noren et al, 1989;Wang et al, 2001). The significance of these methodologies is that by incorporation of nonnatural amino acid residues proteins or peptides can be modulated to have enhanced metabolic and/or thermal stability and/or increased activity .…”

“…It is also possible to assign four base-pair codons as a signal for the analogue incporporation (Magliery et al, 2001). The first sitedirected amino acid incorporation methods relied on chemically acylated tRNAs, which were added to the in vitro translation systems (Noren et al, 1989). Subsequently, through microinjection of aminoacylated tRNAs, membrane proteins could be tagged in vivo with unnatural amino acids in Xenopus oocytes (Nowak et al, 1995).…”

A General Strategy to Access Structural Information at Atomic Resolution in Polyglutamine Homorepeats

“…Consequently, this approach paves the way for investigation of the structural basis of the pathological threshold found in at least ten different poly‐Q diseases. The development of other orthogonal tRNA/synthetase pairs or the use of established synthetic tools, such as flexizyme25 or chemical aminoacylation,17 to load nonsense tRNA with natural amino acids could extend the described strategy for the exploration of other LCRs. In this way, this important but structurally elusive family of proteins can be investigated at high resolution to decipher the structural basis of crucial biological processes and to guide drug design.…”

Modern Synthetic Avenues for the Preparation of Functional Fluorophores

“…Similar chemical transformations have been applied to peptide‐based structures, after the pioneering work from the Schultz group on the genetic code expansion technology 66. Lemke and co‐workers described a set of strained dienophilic unnatural amino acids that could be incorporated into proteins through suppression of the amber stop codon 67.…”