PMID:12021273
| Citation |
Long, JC, DeLeon-Rangel, J and Vik, SB (2002) Characterization of the first cytoplasmic loop of subunit a of the Escherichia coli ATP synthase by surface labeling, cross-linking, and mutagenesis. J. Biol. Chem. 277:27288-93 |
|---|---|
| Abstract |
The first cytoplasmic loop of subunit a of the Escherichia coli ATP synthase has been analyzed by cysteine substitution mutagenesis. 13 of the 26 residues tested were found to be accessible to the reaction with 3-(N-maleimidylpropionyl)-biocytin. The other 13 residues predominantly found in the central region of the polypeptide chain between the two transmembrane spans were more resistant to labeling by 3-(N-maleimidylpropionyl)-biocytin while in membrane vesicle preparations. This region of subunit a contains a conserved residue Glu-80, which when mutated to lysine resulted in a significant loss of ATP-driven proton translocation. Other substitutions including glutamine, alanine, and leucine were much less detrimental to function. Cross-linking studies with a photoactive cross-linking reagent were carried out. One mutant, K74C, was found to generate distinct cross-links to subunit b, and the cross-linking had little effect on proton translocation. The results indicate that the first transmembrane span (residues 40-64) of subunit a is probably near one or both of the b subunits and that a less accessible region of the first cytoplasmic loop (residues 75-90) is probably near the cytoplasmic surface, perhaps in contact with b subunits. |
| Links |
PubMed Online version:10.1074/jbc.M202118200 |
| Keywords |
Adenosine Triphosphate/metabolism; Alanine/chemistry; Amino Acid Sequence; Bacterial Proton-Translocating ATPases/chemistry; Bacterial Proton-Translocating ATPases/metabolism; Cross-Linking Reagents/pharmacology; Cysteine/chemistry; Cytoplasm/metabolism; Escherichia coli/metabolism; Glutamine/chemistry; Immunoblotting; Leucine/chemistry; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Plasmids/metabolism; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Spectrometry, Fluorescence; Time Factors |
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