Incorporation of Nonnatural Amino Acids Into Proteins

Hendrickson, Tamara L.; Crécy‐Lagard, Valérie de; Schimmel, Paul

doi:10.1146/annurev.biochem.73.012803.092429

Cited by 235 publications

(148 citation statements)

References 178 publications

(116 reference statements)

Supporting

Mentioning

145

Contrasting

Unclassified

Order By: Relevance

“…This is interesting fundamentally as well as in the context of the method presented here. Examples of incorporation of unnatural amino acids into proteins by the unmodified endogenous translation machinery are rare 33 and usually confined to bacteria, and many are based on the particular promiscuity of bacterial MetRS 34,35 . Other aaRSs are more stringent, and replacement of natural amino acids is confined to instances involving isosteric unnatural amino acids such as fluorinated derivatives of leucine, phenylalanine and tryptophan 36 .…”

Section: Discussionmentioning

confidence: 99%

Photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells

2005

View full text Add to dashboard Cite

Protein-protein interactions are the key to organizing cellular processes in space and time. The only direct way to identify such interactions in their cellular environment is by photo-crosslinking. Here we present a new strategy for photo-cross-linking proteins in living cells. We designed two new photoactivatable amino acids that we termed photo-methionine and photo-leucine based on their structures and properties closely resembling the natural amino acids methionine and leucine, respectively. This similarity allows them to escape the stringent identity control mechanisms during protein synthesis and be incorporated into proteins by the unmodified mammalian translation machinery. Activation by ultraviolet light induces covalent cross-linking of the interacting proteins, which can be detected with high specificity by simple western blotting. Applying this technology to membrane protein complexes, we discovered a previously unknown direct interaction of the progesterone-binding membrane protein PGRMC1 with Insig-1, a key regulator of cholesterol homeostasis.

show abstract

Section: Discussionmentioning

confidence: 99%

Photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells

2005

View full text Add to dashboard Cite

show abstract

“…Recent efforts aimed at producing proteins with specifically modified residues have provided many examples of how the genetic code of Bacteria and Eucarya can be modulated by laboratory mutation of canonical aminoacyl-tRNA synthetase and cognate tRNA pairs to accept unnatural amino acids (18)(19)(20). In one case, an additional mutant biosynthetic enzyme enabled autogenous biosynthesis and genetic encoding of aminophenylalanine in E. coli (26).…”

Section: Discussionmentioning

confidence: 99%

“…be a powerful avenue to artificially manipulating genetic codes (18)(19)(20). However, transfer of a ''genetic code expansion cassette'' would be unprecedented in natural gene pools.…”

mentioning

confidence: 99%

A natural genetic code expansion cassette enables transmissible biosynthesis and genetic encoding of pyrrolysine

Longstaff

Larue

Faust

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Pyrrolysine has entered natural genetic codes by the translation of UAG, a canonical stop codon. UAG translation as pyrrolysine requires the pylT gene product, an amber-decoding tRNA Pyl that is aminoacylated with pyrrolysine by the pyrrolysyl-tRNA synthetase produced from the pylS gene. The pylTS genes form a gene cluster with pylBCD, whose functions have not been investigated. The pylTSBCD gene order is maintained not only in methanogenic Archaea but also in a distantly related Gram-positive Bacterium, indicating past horizontal gene transfer of all five genes. Here we show that lateral transfer of pylTSBCD introduces biosynthesis and genetic encoding of pyrrolysine into a naïve organism. PylS-based assays demonstrated that pyrrolysine was biosynthesized in Escherichia coli expressing pylBCD from Methanosarcina acetivorans. Production of pyrrolysine did not require tRNA Pyl or PylS. However, when pylTSBCD were coexpressed with mtmB1, encoding the methanogen monomethylamine methyltransferase, UAG was translated as pyrrolysine to produce recombinant monomethylamine methyltransferase. Expression of pylTSBCD also suppressed an amber codon introduced into the E. coli uidA gene. Strains lacking one of the pylBCD genes did not produce pyrrolysine or translate UAG as pyrrolysine. These results indicated that pylBCD gene products biosynthesize pyrrolysine using metabolites common to Bacteria and Archaea and, furthermore, that the pyl gene cluster represents a ''genetic code expansion cassette,'' previously unprecedented in natural organisms, whose transfer allows an existing codon to be translated as a novel endogenously synthesized free amino acid. Analogous cassettes may have served similar functions for other amino acids during the evolutionary expansion of the canonical genetic code.T ranslated in-frame amber (TAG/UAG) codons in the genes encoding methylamine methyltransferases from Methanosarcina barkeri (1-3) were early clues to the existence of pyrrolysine as the 22nd genetically encoded amino acid from nature. Crystallography of MtmB1, the monomethylamine methyltransferase, demonstrated that the UAG codon of the encoding mtmB1 gene corresponds to pyrrolysine, whose structure was proposed as lysine with N in amide linkage with the D-isomer of 4-methyl-pyrroline-5-carboxylate (4, 5). This structure is consistent with the UAG-encoded residue's mass in three distinct methylamine methyltransferases (6).Near the mtmB1 gene in Methanosarcina spp. are the pyl genes (ref. 7; Fig. 1). Two of these genes are known to underlie the genetic encoding of pyrrolysine. The pylT encodes tRNA Pyl , whose CUA anticodon complements the UAG codon corresponding to pyrrolysine. Deletion of the pyl gene promoter and pylT leads to loss of pyrrolysine-dependent monomethylamine methyltransferase and methylamine metabolism (8). The pylS gene encodes the pyrrolysyl-tRNA synthetase, PylS, which charges tRNA Pyl with chemically synthesized pyrrolysine and further carries out a pyrrolysine-dependent ATP:pyrophosphate exchange reaction (5, 9-11)...

show abstract

“…The use of Diels-Alder reactions in protein science may appear paradoxical: the conditions in which these reactions are traditionally carried out during organic synthethic procedures (organic solvents, heating, use of catalysts) are contradictory with the type of reactions which are applied for protein chemistry (aqueous media, room temperature). [1][2][3][4][5] An important objective of the biomedical science is to understand the molecular basis of proteins biological function. Since the explosive success of the genome-sequencing projects, this goal has been dramatically increased as hundreds of thousands of new proteins have been revealed, but only as predicted sequence data.…”

Section: Introductionmentioning

confidence: 99%