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 aci d site specifically into a protein in response to an amber codon intro duced at the corresponding position in the protein's DNA sequence. Usi ng this method, precise changes within a protein can be made to addres s detailed structure-function questions. A series of fluorinated tyros ine analogues and linear, branched, and cyclic hydrophobic amino acids have been used to determine the impact of hydrogen bonding and hydrop hobic packing, respectively, on protein stability. Glutamate analogues and conformationally restricted amino acids have been used to probe t he mechanisms of staphylococcal nuclease and ras. In addition, this te chnique has been used to construct photocaged proteins and proteins co ntaining photoaffinity labels, spin labels, and isotopic labels at spe cific positions in the protein sequence suitable for biophysical studi es.