Introduction of reactive cysteine residues in the epsilon subunit of Escherichia coli F1 ATPase, modification of these sites with tetrafluorophenyl azide-maleimides, and examination of changes in the binding of the epsilon subunit when different nucleotides are in catalytic sites.
Cysteine residues have been exchanged for serine residues at positions 10 and 108 in the epsilon subunit of the Escherichia coli F1 ATPase by site-directed mutagenesis to create two mutants, epsilon-S10C and epsilon-S108C. These two mutants and wild-type enzyme were reacted with [14C]N-ethylmaleimide (NEM) to examine the solvent accessibility of Cys residues and with novel photoactivated cross-linkers, tetrafluorophenyl azide-maleimides (TFPAM's), to examine near-neighbor relationships of subunits. In native wild-type F1 ATPase, NEM reacted with alpha subunits at a maximal level of 1 mol/mol of enzyme (1 mol/3 alpha subunits) and with the delta subunit at 1 mol/mol of enzyme; other subunits were not labeled by the reagent. In the mutants epsilon-S10C and epsilon-S108C, Cys10 and Cys108, respectively, were also labeled by NEM, indicating that these are surface residues. Reaction of wild-type enzyme with TFPAM's gave cross-linking of the delta subunit to both alpha and beta subunits. Reaction of the mutants with TFPAM's also cross-linked delta to alpha and beta and in addition formed covalent links between Cys10 of the epsilon subunit and the gamma subunit and between Cys108 of the epsilon subunit and the alpha subunit. The yield of cross-linking between sites on epsilon and other subunits depended on the nucleotide conditions used; this was not the case for delta-alpha or delta-beta cross-linked products. In the presence of ATP+EDTA the yield of cross-linking between epsilon-Cys10 and gamma was high (close to 50%) while the yield of epsilon-Cys108 and alpha was low (around 10%).(ABSTRACT TRUNCATED AT 250 WORDS)
Aggeler
R
R
Institute of Molecular Biology, University of Oregon, Eugene 97403.
Chicas-Cruz
K
K
Cai
S X
SX
Keana
J F
JF
Capaldi
R A
RA
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001386:Azides; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000737:chemistry*; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D005033:Ethylmaleimide; D005464:Fluorobenzenes; D008274:Magnesium / Q000494:pharmacology; D008301:Maleimides; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010782:Photolysis; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D001386:Azides; D003432:Cross-Linking Reagents; D005464:Fluorobenzenes; D008301:Maleimides; C073711:N-(4-azido-2,3,5,6-tetrafluorobenzyl)-3-maleimidopropionamide; C073709:N-(4-azido-2,3,5,6-tetrafluorobenzyl)-6-maleimidohexanamide; D000255:Adenosine Triphosphate; D004492:Edetic Acid; D006180:Proton-Translocating ATPases; D008274:Magnesium; D003545:Cysteine; D005033:Ethylmaleimide