Helix packing in subunit a of the Escherichia coli ATP synthase as determined by chemical labeling and proteolysis of the cysteine-substituted protein.
Subunit a of the Escherichia coli ATP synthase is thought to control access of protons to the ring of c subunits during proton-driven ATP synthesis. In this study, the surface exposure of subunit a in the periplasm has been examined using 3-N-maleimidyl-propionyl biocytin labeling in cells permeabilized by polymyxin B nonapeptide, and the helix packing at the periplasmic surface has been probed by metal-chelate mediated proteolysis. Eighteen residues between 119 and 146 were changed individually to cysteine and tested for accessibility. Positions labeled included D124 and D146, indicating a periplasmic loop of at least 23 amino acids. Residues near the ends of the transmembrane spans were tested with 5-(alpha-bromoacetamido)-1,10-phenanthroline-copper for chemical proteolysis. Only residues W241C and D44C, and to a lesser extent I43C, led to proteolytic fragments after oxidation. The fragments were sized by comparison with molecular weight standards generated by Factor Xa sites engineered into subunit a. Fragments were detected by immunoblotting using an engineered HA epitope at the carboxyl-terminal end of subunit a. The results indicated that both transmembrane span 5 (W241) and transmembrane span 1 (D44) are close to transmembrane span 2.
Zhang
Di
D
Department of Biological Sciences, Southern Methodist University, Dallas, Texas 75275-0376, USA.
Vik
Steven B
SB
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D003300:Copper; D003545:Cysteine / Q000235:genetics; D010450:Endopeptidases / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D006868:Hydrolysis; D008239:Lysine / Q000031:analogs & derivatives*; D008301:Maleimides; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009942:Organometallic Compounds; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D010618:Phenanthrolines; D017433:Protein Structure, Secondary / Q000235:genetics; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics
C095824:5-(bromoacetamido)-1,10-phenanthroline copper complex; D029968:Escherichia coli Proteins; D008301:Maleimides; D008565:Membrane Proteins; D009942:Organometallic Compounds; D010446:Peptide Fragments; D010618:Phenanthrolines; D021122:Protein Subunits; D003300:Copper; C047272:3-(N-maleimidopropionyl)biocytin; D010450:Endopeptidases; D006180:Proton-Translocating ATPases; D008239:Lysine; D003545:Cysteine