title: Site-specific, photochemical proteolysis applied to ion channels in vivo.
abstract: A method for site-specific, nitrobenzyl-induced photochemical proteolysis of diverse proteins expressed in living cells has been developed based on the chemistry of the unnatural amino acid (2-nitrophenyl)glycine (Npg). Using the in vivo nonsense codon suppression method for incorporating unnatural amino acids into proteins expressed in Xenopus oocytes, Npg has been incorporated into two ion channels: the Drosophila Shaker B K+ channel and the nicotinic acetylcholine receptor. Functional studies in vivo show that irradiation of proteins containing an Npg residue does lead to peptide backbone cleavage at the site of the novel residue. Using this method, evidence is obtained for an essential functional role of the "signature" Cys128-Cys142 disulfide loop of the nAChR alpha subunit.
authors.affiliation: Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.
authors.forename: P M
authors.initials: PM
authors.lastname: England
authors.forename: H A
authors.initials: HA
authors.lastname: Lester
authors.forename: N
authors.initials: N
authors.lastname: Davidson
authors.forename: D A
authors.initials: DA
authors.lastname: Dougherty
mesh_terms: D000818:Animals; D004330:Drosophila; D029721:Drosophila Proteins; D006868:Hydrolysis; D016296:Mutagenesis; D009865:Oocytes / Q000378:metabolism / Q000528:radiation effects; D010777:Photochemistry; D015221:Potassium Channels / Q000235:genetics / Q000378:metabolism* / Q000528:radiation effects; D011978:Receptors, Nicotinic / Q000235:genetics; D051637:Shaker Superfamily of Potassium Channels; D014982:Xenopus laevis
chemical_list: D029721:Drosophila Proteins; D015221:Potassium Channels; D011978:Receptors, Nicotinic; C496929:Sh protein, Drosophila; D051637:Shaker Superfamily of Potassium Channels