Escherichia coli H+-ATPase. Glutamic acid 185 in beta subunit is essential for its structure and assembly.
The uncD gene for the beta subunit of Escherichia coli H+-ATPase was cloned downstream of the lac promoter and mutagenized (Glu-185----Gln or Lys) by an oligonucleotide-directed procedure. The recombinant plasmid was introduced into a strain in which the unc operon for subunits of H+-ATPase was deleted. The wild-type or mutant beta subunit synthesized amounted to about 10% total cell protein and was mainly found in the cytoplasmic fraction. These subunits could be purified to almost homogeneity by conventional procedures. The wild-type and two mutant beta subunits had essentially the same Kd values for 8-anilinonaphthalene-1-sulfonate, aurovertin, and ATP, although the fluorescence intensities of 8-anilinonaphthalene-1-sulfonate and aurovertin were significantly less when bound to the two mutant beta subunits than when bound to the wild-type subunit. The three beta subunits showed essentially the same circular dichroism spectra, indicating alpha-helical contents of about 16-18%. Thus, the mutations did not cause marked change of the secondary structure of the subunit. However, measurements of theta 208 during linear increase in temperature suggested that replacement of Glu-185 by Gln or Lys slightly changed the stability of the secondary structure. Only trace amounts of alpha beta gamma complexes could be reconstituted using the two mutant beta subunits. These results suggest that Glu-185 or the region in its vicinity may be essential for subunit assembly. The methods developed in this study should be useful for further studies on the beta subunit.
D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D005971:Glutamates; D018698:Glutamic Acid; D046911:Macromolecular Substances; D009154:Mutation; D010957:Plasmids; D011487:Protein Conformation; D011489:Protein Denaturation; D006180:Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism
D005971:Glutamates; D046911:Macromolecular Substances; D018698:Glutamic Acid; D006180:Proton-Translocating ATPases