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Subunit a of the E. coli ATP synthase: reconstitution and high resolution NMR with protein purified in a mixed polarity solvent
Author(s) -
Dmitriev Oleg Y.,
Altendorf Karlheinz,
Fillingame Robert H.
Publication year - 2004
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(03)01360-7
Subject(s) - protein subunit , chemistry , atp synthase , solvent , atp synthase gamma subunit , nuclear magnetic resonance spectroscopy , proton nmr , chloroform , escherichia coli , chromatography , aqueous solution , membrane , biochemistry , enzyme , stereochemistry , organic chemistry , atpase , atp hydrolysis , gene
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.