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Gene Synthesis, Expression, and Mutagenesis of the Blue Copper Proteins Azurin and Plastocyanin

Chang, Thomas K. and Iverson, Sheila A. and Rodrigues, Clyde G. and Kiser, Cynthia N. and Lew, Agnes Y. C. and Germanas, Juris P. and Richards, John H. (1991) Gene Synthesis, Expression, and Mutagenesis of the Blue Copper Proteins Azurin and Plastocyanin. Proceedings of the National Academy of Sciences of the United States of America, 88 (4). pp. 1325-1329. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:CHApnas91

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Abstract

Genes for the blue copper proteins Populus nigra var. italica plastocyanin and Pseudomonas aeruginosa azurin have been constructed by a stepwise procedure. The leader sequence for azurin has been placed before the genes directing plastocyanin and azurin transport to the periplasmic space when the genes are expressed in Escherichia coli. Site-saturation mutagenesis has been used to alter two copperbinding residues of azurin (Met-121 and His-46) and Met-92 of plastocyanin. While the plastocyanin mutants do not appear to bind copper, the azurin variants all bind copper and show characteristic type I blue copper centers. In particular, the electronic spectra reflect the dominance of the charge transfer interaction between copper and the thiolate of Cys-112, being relatively insensitive to changes in Met-121 or His-46. In contrast, removal of Met-121 appreciably alters the EPR spectra of the mutants, although, to a first order, the spectra of all mutants are themselves similar, suggesting a more distorted geometry around copper in the mutants than in the wild type.


Item Type:Article
Additional Information:Copyright © 1991 by National Academy of Sciences. Communicated by Harry B. Gray, October 31, 1990. We gratefully acknowledge early encouragement for this work by I. Pecht and the continuing enthusiasm of H.B. Gray and Angel J. Di Bilio for determining the EPR spectra. This work was supported by National Institutes of Health Grant GM16424, National Science Foundation Grant CHE-8814222, and National Institutes of Health Grant DK19038. This is contribution 8218 from Division of Chemistry and Chemical Engineering of the California Institute of Technology. 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:copper center; synthetic gene
Record Number:CaltechAUTHORS:CHApnas91
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:CHApnas91
Alternative URL:http://www.pnas.org/cgi/content/abstract/88/4/1325
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:9910
Collection:CaltechAUTHORS
Deposited By: Archive Administrator
Deposited On:26 Mar 2008
Last Modified:26 Dec 2012 09:54

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