The genetic incorporation of a distance probe into proteins in Escherichia coli.
The unnatural amino acid p-nitrophenylalanine (pNO2-Phe) was genetically introduced into proteins in Escherichia coli in response to the amber nonsense codon with high fidelity and efficiency by means of an evolved tRNA/aminoacyl-tRNA synthetase pair from Methanocuccus jannaschii. It was shown that pNO2-Phe efficiently quenches the intrinsic fluorescence of Trp in a distance-dependent manner in a model GCN4 basic region leucine zipper (bZIP) protein. Thus, the pNO2-Phe/Trp pair should be a useful biophysical probe of protein structure and function.
Tsao
Meng-Lin
ML
Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Summerer
Daniel
D
Ryu
Youngha
Y
Schultz
Peter G
PG
D000595:Amino Acid Sequence; D050976:Basic-Leucine Zipper Transcription Factors / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D017017:Methanococcus / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000096:biosynthesis / Q000235:genetics*; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000235:genetics / Q000378:metabolism; D012365:RNA, Transfer, Tyr / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014157:Transcription Factors / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014448:Tyrosine-tRNA Ligase / Q000235:genetics / Q000378:metabolism
D050976:Basic-Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C501391:GCN4 protein, S cerevisiae; D050505:Mutant Proteins; D012365:RNA, Transfer, Tyr; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C040239:4-nitrophenylalanine; D010649:Phenylalanine; D014364:Tryptophan; D014448:Tyrosine-tRNA Ligase