Mutational analysis of G protein alpha subunit G(o) alpha expressed in Escherichia coli.
G protein-mediated signal transduction is dependent on alpha subunit interactions with beta gamma 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 G(o) alpha. Random mutations were generated by polymerase chain reaction in the amino-terminal 56 amino acids of G(o) alpha. Guanine nucleotide binding properties of the mutants were assayed in situ and in crude extracts of recombinant E. coli. beta gamma interactions were assayed by pertussis toxin mediated ADP-ribosylation. Efficacy of the screening procedures was evaluated by studying properties of wild-type G(o) alpha 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.
Slepak
V Z
VZ
Biology Division, California Institute of Technology, Pasadena 91125.
Wilkie
T M
TM
Simon
M I
MI
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D037342:Pertussis Toxin; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D004247:DNA; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins