title: Cell-free synthesis of membrane subunits of ATP synthase in phospholipid bicelles: NMR shows subunit a fold similar to the protein in the cell membrane.
abstract: NMR structure determination of large membrane proteins is hampered by broad spectral lines, overlap, and ambiguity of signal assignment. Chemical shift and NOE assignment can be facilitated by amino acid selective isotope labeling in cell-free protein synthesis system. However, many biological detergents are incompatible with the cell-free synthesis, and membrane proteins often have to be synthesized in an insoluble form. We report cell-free synthesis of subunits a and c of the proton channel of Escherichia coli ATP synthase in a soluble form in a mixture of phosphatidylcholine derivatives. In comparison, subunit a was purified from the cell-free system and from the bacterial cell membranes. NMR spectra of both preparations were similar, indicating that our procedure for cell-free synthesis produces protein structurally similar to that prepared from the cell membranes.
authors.affiliation: Department of Biochemistry, University of Saskatchewan, 107 Wiggins Road, Saskatoon, SK, Canada.
authors.forename: Eva-Maria E
authors.initials: EM
authors.lastname: Uhlemann
authors.forename: Hannah E
authors.initials: HE
authors.lastname: Pierson
authors.forename: Robert H
authors.initials: RH
authors.lastname: Fillingame
authors.forename: Oleg Y
authors.initials: OY
authors.lastname: Dmitriev
mesh_terms: D025243:Bacterial Proton-Translocating ATPases / Q000737:chemistry / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D002474:Cell-Free System / Q000201:enzymology / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry; D008823:Micelles; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010743:Phospholipids / Q000737:chemistry / Q000494:pharmacology; D017510:Protein Folding; D055503:Protein Multimerization / Q000502:physiology; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000096:biosynthesis / Q000737:chemistry; D006180:Proton-Translocating ATPases / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D008823:Micelles; D010743:Phospholipids; D021122:Protein Subunits; C074429:atpE protein, E coli; D025243:Bacterial Proton-Translocating ATPases; D006180:Proton-Translocating ATPases; C074428:atpB protein, E coli