Mutational analysis of G protein α subunit Goα expressed in Escherichia coli

Vladlen Z. Slepak, Thomas M. Wilkie, Melvin I. Simon

Research output: Contribution to journalArticle

31 Citations (Scopus)

Abstract

G protein-mediated signal transduction is dependent on α subunit interactions with βγ 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 Goa. Random mutations were generated by polymerase chain reaction in the amino-terminal 56 amino acids of Goa. Guanine nucleotide binding properties of the mutants were assayed in situ and in crude extracts of recombinant E. coli. βγ 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 βγ 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.

Original languageEnglish (US)
Pages (from-to)1414-1423
Number of pages10
JournalJournal of Biological Chemistry
Volume268
Issue number2
StatePublished - Jan 15 1993

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Gi-Go GTP-Binding Protein alpha Subunits
GTP-Binding Proteins
Escherichia coli
Guanine Nucleotides
Adenosine Diphosphate
Magnesium
Mutation
Screening
Bearings (structural)
Guanosine 5'-O-(3-Thiotriphosphate)
Amino Acids
Signal transduction
Mutagenesis
Polymerase chain reaction
Pertussis Toxin
Amino Acid Substitution
Guanosine Triphosphate
Complex Mixtures
Refractory materials
Signal Transduction

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mutational analysis of G protein α subunit Goα expressed in Escherichia coli. / Slepak, Vladlen Z.; Wilkie, Thomas M.; Simon, Melvin I.

In: Journal of Biological Chemistry, Vol. 268, No. 2, 15.01.1993, p. 1414-1423.

Research output: Contribution to journalArticle

Slepak, Vladlen Z. ; Wilkie, Thomas M. ; Simon, Melvin I. / Mutational analysis of G protein α subunit Goα expressed in Escherichia coli. In: Journal of Biological Chemistry. 1993 ; Vol. 268, No. 2. pp. 1414-1423.
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