A new strategy for the site-specific modification of proteins in vivo.
We recently developed a method for genetically incorporating unnatural amino acids site-specifically into proteins expressed in Escherichia coli in response to the amber nonsense codon. Here we describe the selection of an orthogonal tRNA-TyrRS pair that selectively and efficiently incorporates m-acetyl-l-phenylalanine into proteins in E. coli. We demonstrate that proteins containing m-acetyl-l-phenylalanine or p-acetyl-l-phenylalanine can be selectively labeled with hydrazide derivatives not only in vitro but also in living cells. The labeling reactions are selective and in general proceed with yields of >75%. In specific examples, m-acetyl-l-phenylalanine was substituted for Lys7 of the cytoplasmic protein Z domain, and for Arg200 of the outer membrane protein LamB, and the mutant proteins were selectively labeled with a series of fluorescent dyes. The genetic incorporation of a nonproteinogenic "ketone handle" into proteins provides a powerful tool for the introduction of biophysical probes for the structural and functional analysis of proteins in vitro or in vivo.
Zhang
Zhiwen
Z
Department of Chemistry and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Smith
Brian A C
BA
Wang
Lei
L
Brock
Ansgar
A
Cho
Charles
C
Schultz
Peter G
PG
D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005452:Fluoresceins / Q000737:chemistry; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D012343:RNA, Transfer / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics*; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D017931:DNA Primers; D005452:Fluoresceins; D011994:Recombinant Proteins; D014443:Tyrosine; D010649:Phenylalanine; D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases