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Site-specific, photochemical proteolysis applied to ion channels in vivo

England, Pamela M. and Lester, Henry A. and Davidson, Norman and Dougherty, Dennis A. (1997) Site-specific, photochemical proteolysis applied to ion channels in vivo. Proceedings of the National Academy of Sciences of the United States of America, 94 (20). pp. 11025-11030. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:ENGpnas97

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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.


Item Type:Article
Additional Information:Copyright © 1997 by the National Academy of Sciences. Communicated by Peter B. Dervan, California Institute of Technology, Pasadena, CA, July 11, 1997 (received for review April 20, 1997). We thank P. Reinhart, P. Kearney, M. Nowak, and S. Silverman for suggestions and helpful discussions and B. Henkle and H. Li for assistance with oocytes. This work was supported by the National Institutes of Health (NS-34407, NS-11756, and a National Research Service Award to P.M.E.) and the University of California Tobacco-Related Disease Research Program. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Subject Keywords:nicotinic acetylcholine receptor, (2-nitrophenyl)glycine, K+ channel, signature disulfide loop, site-specific, nitrobenzyl-induced photochemical proteolysis, conserved disulfide loop, unnatural amino-acids, potassium channel, binding-site, chemical aminoacylation, photolabile precursors, caged compounds, alpha-subunit, M2 domain
Record Number:CaltechAUTHORS:ENGpnas97
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:ENGpnas97
Alternative URL:http://www.pnas.org/cgi/content/abstract/94/20/11025
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:906
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:07 Nov 2005
Last Modified:14 Nov 2014 19:18

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