title: Functional incorporation of chimeric b subunits into F1Fo ATP synthase.
abstract: F(1)F(o) ATP synthases function by a rotary mechanism. The enzyme's peripheral stalk serves as the stator that holds the F(1) sector and its catalytic sites against the movement of the rotor. In Escherichia coli, the peripheral stalk is a homodimer of identical b subunits, but photosynthetic bacteria have open reading frames for two different b-like subunits thought to form heterodimeric b/b' peripheral stalks. Chimeric b subunit genes have been constructed by substituting sequence from the Thermosynechococcus elongatus b and b' genes in the E. coli uncF gene, encoding the b subunit. The recombinant genes were expressed alone and in combination in the E. coli deletion strain KM2 (Deltab). Although not all of the chimeric subunits were incorporated into F(1)F(o) ATP synthase complexes, plasmids expressing either chimeric b(E39-I86) or b'(E39-I86) were capable of functionally complementing strain KM2 (Deltab). Strains expressing these subunits grew better than cells with smaller chimeric segments, such as those expressing the b'(E39-D53) or b(L54-I86) subunit, indicating intragenic suppression. In general, the chimeric subunits modeled on the T. elongatus b subunit proved to be more stable than the b' subunit in vitro. Coexpression of the b(E39-I86) and b'(E39-I86) subunits in strain KM2 (Deltab) yielded F(1)F(o) complexes containing heterodimeric peripheral stalks composed of both subunits.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32605, USA.
authors.forename: Shane B
authors.initials: SB
authors.lastname: Claggett
authors.forename: Tammy Bohannon
authors.initials: TB
authors.lastname: Grabar
authors.forename: Stanley D
authors.initials: SD
authors.lastname: Dunn
authors.forename: Brian D
authors.initials: BD
authors.lastname: Cain
mesh_terms: D000595:Amino Acid Sequence; D000458:Cyanobacteria / Q000201:enzymology* / Q000235:genetics; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D017353:Gene Deletion; D005816:Genetic Complementation Test; D025261:Mitochondrial Proton-Translocating ATPases / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010957:Plasmids; D021122:Protein Subunits / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D021122:Protein Subunits; D011994:Recombinant Proteins; C474451:F1F0-ATP synthase; D025261:Mitochondrial Proton-Translocating ATPases