Site-directed mutagenesis with an expanded genetic code.
A biosynthetic method has been developed that makes possible the site-specific incorporation of a large number of amino acids and analogues within proteins. In this approach, an amber suppressor tRNA chemically aminoacylated with the desired amino acid incorporates this amino acid site specifically into a protein in response to an amber codon introduced at the corresponding position in the protein's DNA sequence. Using this method, precise changes within a protein can be made to address detailed structure-function questions. A series of fluorinated tyrosine analogues and linear, branched, and cyclic hydrophobic amino acids have been used to determine the impact of hydrogen bonding and hydrophobic packing, respectively, on protein stability. Glutamate analogues and conformationally restricted amino acids have been used to probe the mechanisms of staphylococcal nuclease and ras. In addition, this technique has been used to construct photocaged proteins and proteins containing photoaffinity labels, spin labels, and isotopic labels at specific positions in the protein sequence suitable for biophysical studies.
Mendel
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Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285-0540, USA.
Cornish
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VW
Schultz
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PG
D000596:Amino Acids / Q000737:chemistry; D018388:Codon, Terminator; D004252:DNA Mutational Analysis; D004798:Enzymes / Q000378:metabolism; D005815:Genetic Code; D015335:Molecular Probes; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D015398:Signal Transduction
D000596:Amino Acids; D018388:Codon, Terminator; D004798:Enzymes; D015335:Molecular Probes