A general biosynthetic method has been developed which makes it possible to site-specifically incorporate unnatural amino acids with novel properties into proteins. In this approach the codon encoding the amino acid of interest is replaced with the "blank" nonsense codon UAG by oligonucleotide-directed mutagenesis. A suppressor tRNA that recognizes this codon is generated by run-off transcription and then chemically aminoacylated with the desired unnatural amino acid. Addition of the mutagenized gene and the aminoacylated suppressor tRNA to an in vitro extract capable of supporting protein biosynthesis generates a mutant protein containing the unnatural amino acid at the specified position. This methodology has recently been used to study the stability, specificity, and catalytic properties of a number of proteins. In these studies amino acids and analogues possessing altered hydrogen-bonding, electronic. and steric properties and unique backbone conformations have all been site-specifically incorporated into proteins. In addition, photoactivatable amino acids, isotopically labeled amino acids, and amino acids bearing biophysical probes have been inserted site-specifically . This chemistry increases our ability to carry out detailed physical organic studies on this important class of macromolecules.

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A number of unnatural amino acids and amino acid analogs with modified backbone structures were substituted for alanine-82 in T4 lysozyme. Replacements included alpha,alpha-disubstituted amino acids, N-alkyl amino acids, and lactic acid, an isoelectronic analog of alanine. The effects of these electronic and structural perturbations on the stability of T4 lysozyme were determined. The relatively broad substrate specificity of the Escherichia coli protein biosynthetic machinery suggests that a wide range of backbone and side-chain substitutions can be introduced, allowing a more precise definition of the factors affecting protein stability.

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.