SITE-SPECIFIC, PHOTOCHEMICAL PROTEOLYSIS APPLIED TO ION CHANNELS IN-VIVO

A method for site-specific, nitrobenzyl-induced photochemical proteoly sis of diverse proteins expressed in living cells has been developed b ased on the chemistry of the unnatural amino acid (2-nitrophenyl)glyci ne (Npg). Using the in vivo nonsense codon suppression method for inco rporating unnatural amino acids into proteins expressed in Xenopus ooc ytes, Npg has been incorporated into two ion channels: the Drosophila Shaker B K+ channel and the nicotinic acetylcholine receptor. Function al studies in vivo show that irradiation of proteins containing an Npg residue does lead to peptide backbone cleavage at the site of the nov el residue. Using this method, evidence is obtained for an essential f unctional role of the ''signature'' Cys128-Cys142 disulfide loop of th e nAChR alpha subunit.