Molecular architecture of the c-subunit oligomer in the membrane domain of F-ATPases probed by tryptophan substitution mutagenesis.
Subunit c of the proton-transporting ATP synthase of Escherichia coli forms an oligomeric complex in the membrane domain that functions in transmembrane proton conduction. In order to gain some insight into the architecture of this oligomeric complex, the transmembrane region in the C-terminal membrane-spanning segment was analysed by a site-directed mutagenesis approach. Tryptophan substitution of consecutive residues in positions 61 to 72 of subunit c was used to identify residues oriented towards a helix-helix surface or an accessible phase in the oligomeric complex. Mutants were analysed in functional assays of ATP hydrolysis, ATP synthesis and ATP-dependent proton transport. Function was disrupted according to a pattern that identified inter- and intramolecular contacts in the c-subunit oligomer. Screening experiments on minimal medium support the helix-helix contacts found in the functional assays. The results add strong constraints to the potential orientation of the monomers in the oligomeric complex and are discussed against the background of different structural models that have been proposed for the c-subunit oligomer.
Biochemie der Pflanzen, Heinrich-Heine Universität Düssseldorf, Universitätsstr.1, Düsseldorf, 40225, Germany. georg.groth@uni-duesseldorf.de
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Groth
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Tilg
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Schirwitz
D000255:Adenosine Triphosphate / Q000096:biosynthesis; D019943:Amino Acid Substitution; D001483:Base Sequence; D003470:Culture Media; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011522:Protons; D014364:Tryptophan / Q000737:chemistry
D003470:Culture Media; D004269:DNA, Bacterial; D011522:Protons; D014364:Tryptophan; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases