Slepak, Vladlen Z. and Wilkie, Thomas M. and Simon, Melvin I. (1993) Mutational Analysis of G Protein α Subunit G_oα Expressed in Escherichia coli. Journal of Biological Chemistry, 268 (2). pp. 1414-1423. ISSN 0021-9258 http://resolver.caltech.edu/CaltechAUTHORS:20120307-154214152
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G protein-mediated signal transduction is dependent on alpha subunit interactions with βy subunits, receptors, effectors, magnesium ions, and guanine nucleotides. The interdependence of these interactions can be probed by mutational analysis. We developed large scale screening procedures in recombinant Escherichia coli to identify and characterize novel mutations in Goα. Random mutations were generated by polymerase chain reaction in the amino-terminal 56 amino acids of Goα. Guanine nucleotide binding properties of the mutants were assayed in situ and in crude extracts of recombinant E. coli. βy interactions were assayed by pertussis toxin mediated ADP-ribosylation. Efficacy of the screening procedures was evaluated by studying properties of wild-type Goα and site-directed mutations that were characterized previously in other G proteins. Several novel mutants with altered GTP binding characteristics and reduced ability to interact with beta gamma had been isolated from the randomly generated mutant library. ADP-ribosylation of mutants R10G, K21N, and K35E was significantly reduced, whereas two of the mutants bearing multiple amino acid substitutions were refractory to modification. Mutant K35E also exhibited reduced affinity to guanosine 5'-(3-O-thio)triphosphate at submicromolar concentrations of magnesium. These experiments demonstrate the feasibility of using large scale random mutagenesis in the studies of G protein function.
|Additional Information:||© 1993 American Society for Biochemistry and Molecular Biology Inc. Received for publication, August 17, 1992. This work was supported by National Institutes of Health Grant GM 34236. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. We thank Drs. James Hurley, Ray Mosteller, and Patrick Casey for providing preparations of G protein βy subunit complex. We also thank Dr. Maurine Linder for sharing the plasmids pBB131 and NpT7-G_i1 and Dr. Allen Spiegel and Susan Mumby for the antibodies. We thank Bruce W. Birren for reading the manuscript.|
|Official Citation:||Mutational analysis of G protein α subunit Goα expressed in Escherichia coli. V Z Slepak, T M Wilkie, and M I Simon J. Biol. Chem. 1993 268: 1414-1423|
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|Deposited By:||Ruth Sustaita|
|Deposited On:||12 Mar 2012 20:56|
|Last Modified:||12 Mar 2012 20:56|
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