The b subunits in the peripheral stalk of F1F0 ATP synthase preferentially adopt an offset relationship.
The peripheral stalk of F1F0 ATP synthase is essential for the binding of F1 to FO and for proper transfer of energy between the two sectors of the enzyme. The peripheral stalk of Escherichia coli is composed of a dimer of identical b subunits. In contrast, photosynthetic organisms express two b-like genes that form a heterodimeric peripheral stalk. Previously we generated chimeric peripheral stalks in which a portion of the tether and dimerization domains of the E. coli b subunits were replaced with homologous sequences from the b and b' subunits of Thermosynechococcus elongatus (Claggett, S. B., Grabar, T. B., Dunn, S. D., and Cain, B. D. (2007) J. Bacteriol. 189, 5463-5471). The spatial arrangement of the chimeric b and b' subunits, abbreviated Tb and Tb', has been investigated by Cu2+-mediated disulfide cross-link formation. Disulfide formation was studied both in soluble model polypeptides and between full-length subunits within intact functional F1F0 ATP synthase complexes. In both cases, disulfides were preferentially formed between TbA83C and Tb'A90C, indicating the existence of a staggered relationship between helices of the two chimeric subunits. Even under stringent conditions rapid formation of disulfides between these positions occurred. Importantly, formation of this cross-link had no detectable effect on ATP-driven proton pumping, indicating that the staggered conformation is compatible with normal enzymatic activity. Under less stringent reaction conditions, it was also possible to detect b subunits cross-linked through identical positions, suggesting that an in-register, nonstaggered parallel conformation may also exist.
From the Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32605.
Shane B
SB
Claggett
From the Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32605.
Mac O'Neil
MO
Plancher
Department of Biochemistry, University of Western Ontario, London, Ontario N6A 5C1, Canada.
Stanley D
SD
Dunn
From the Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, Florida 32605. Electronic address: bcain@biochem.med.ufl.edu.
Brian D
BD
Cain
D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D025243:Bacterial Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000378:metabolism; D000458:Cyanobacteria / Q000201:enzymology*; D003545:Cysteine / Q000235:genetics; D019281:Dimerization; D004220:Disulfides / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
D003432:Cross-Linking Reagents; D004220:Disulfides; D021122:Protein Subunits; D000255:Adenosine Triphosphate; D025243:Bacterial Proton-Translocating ATPases; D003545:Cysteine