PMID:12842903
| Citation |
Grabar, TB and Cain, BD (2003) Integration of b subunits of unequal lengths into F1F0-ATP synthase. J. Biol. Chem. 278:34751-6 |
|---|---|
| Abstract |
In Escherichia coli the peripheral stalk of F1F0-ATP synthase consists of a parallel dimer of identical b subunits. However, the length of the two b subunits need not be fixed. This led us to ask whether it is possible for two b subunits of unequal length to dimerize in a functional enzyme complex. A two-plasmid expression system has been developed that directs production of b subunits of unequal lengths in the same cell. Two b subunits differing in length have been expressed with either a histidine or V5 epitope tag to facilitate nickel-affinity resin purification (Ni-resin) and Western blot analysis. The epitope tags did not materially affect enzyme function. The system allowed us to determine whether the different b subunits segregate to form homodimers or, conversely, whether a heterodimer consisting of both the shortened and lengthened b subunits can occur in an intact enzyme complex. Experiments expressing different b subunits lengthened and shortened by up to 7 amino acids were detected in the same enzyme complex. The V5-tagged b subunit shortened by 7 amino acids (b Delta 7-V5) was detected in Ni-resin-purified membrane preparations only when coexpressed with a histidine-tagged b subunit in the same cell. The results demonstrate that the enzyme complex can tolerate a size difference between the two b subunits of up to 14 amino acids. Moreover, the experiments demonstrated the feasibility of constructing enzyme complexes with non-identical b subunits that will be valuable for research requiring specific chemical modification of a single b subunit. |
| Links |
PubMed Online version:10.1074/jbc.M303361200 |
| Keywords |
Base Sequence; Cell Membrane/enzymology; DNA Primers; Epitopes/chemistry; Kinetics; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Plasmids; Protein Conformation; Protein Subunits; Proton-Translocating ATPases/biosynthesis; Proton-Translocating ATPases/chemistry; Proton-Translocating ATPases/genetics; Recombinant Proteins/biosynthesis; Restriction Mapping; Sequence Deletion |
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