Subunit A of the E. coli ATP synthase: reconstitution and high resolution NMR with protein purified in a mixed polarity solvent.
Subunit a of the Escherichia coli ATP synthase, a 30 kDa integral membrane protein, was purified to homogeneity by a novel procedure incorporating selective extraction into a monophasic mixture of chloroform, methanol and water, followed by Ni-NTA chromatography in the mixed solvent. Pure subunit a was reconstituted with subunits b and c and phospholipids to form a functional proton-translocating unit. Nuclear magnetic resonance (NMR) spectra of the pure subunit a in the mixed solvent show good chemical shift dispersion and demonstrate the potential of the solvent mixture for NMR studies of the large membrane proteins that are currently intractable in aqueous detergent solutions.
Dmitriev
Oleg Y
OY
Department of Biomolecular Chemistry, University of Wisconsin Medical School, 1300 University Avenue, Madison, WI 53706-1532, USA.
Altendorf
Karlheinz
K
Fillingame
Robert H
RH
D000595:Amino Acid Sequence; D025243:Bacterial Proton-Translocating ATPases; D002725:Chloroform / Q000737:chemistry; D002847:Chromatography, Agarose / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification; D000432:Methanol / Q000737:chemistry; D025261:Mitochondrial Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011510:Proteolipids / Q000378:metabolism; D011522:Protons; D012997:Solvents / Q000737:chemistry; D014867:Water / Q000737:chemistry
D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D021122:Protein Subunits; D011510:Proteolipids; D011522:Protons; D012997:Solvents; D014867:Water; D002725:Chloroform; D025243:Bacterial Proton-Translocating ATPases; D025261:Mitochondrial Proton-Translocating ATPases; C074428:atpB protein, E coli; D000432:Methanol