Location of conserved residue histidine-38 of the epsilon subunit of Escherichia coli ATP synthase.
The function and location of residue His-38 of the epsilon subunit of the Escherichia coli F1-ATPase were investigated. His-38 was replaced by glutamine and cysteine through site-directed mutagenesis to produce epsilon H38Q and epsilon H38C, respectively. Both epsilon H38Q and epsilon H38C fulfilled epsilon function in vivo as determined by growth on nonfermentable carbon sources, growth yield on limiting glucose, and recovery of cells from energy starvation conditions. epsilon H38Q and epsilon H38C were purified and studied in vitro. Pure epsilon H38C reacted rapidly with Ellman's reagent, indicating a surface location of the introduced cysteine. epsilon H38C which had been reconstituted with epsilon-depleted F1-ATPase could be linked specifically to the gamma subunit using two different heterobifunctional sulfhydril-reactive/photoreactive crosslinking agents, indicating that residue 38 lies near gamma. The mutated epsilon subunits were unaltered in their ability to inhibit epsilon-depleted F1-ATPase in vitro, even after modification of epsilon H38C with the bulky reagents fluorescein maleimide and N-(1-anilinonaphthyl-4)maleimide. It seems unlikely, therefore, that residue His-38 of epsilon interacts directly with gamma. Both the epsilon H38Q and epsilon H38C mutations reduced the recognition of epsilon by monoclonal antibody epsilon-1, but recognition of epsilon H38C was not further reduced by reaction with fluorescein maleimide. These results imply that residue 38 is not directly part of the epsilon-1 epitope, but plays a role in its formation.
Skakoon
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Department of Biochemistry, University of Western Ontario, London, Canada.
Dunn
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D000911:Antibodies, Monoclonal; D001483:Base Sequence; D017124:Conserved Sequence; D003432:Cross-Linking Reagents; D004228:Dithionitrobenzoic Acid; D000939:Epitopes / Q000276:immunology; D004926:Escherichia coli / Q000201:enzymology*; D006639:Histidine / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010777:Photochemistry; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D000939:Epitopes; D006639:Histidine; D004228:Dithionitrobenzoic Acid; D006180:Proton-Translocating ATPases