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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
Author(s) -
Uhlemann EvaMaria E.,
Pierson Hannah E.,
Fillingame Robert H.,
Dmitriev Oleg Y.
Publication year - 2012
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.2014
Subject(s) - atp synthase , biochemistry , membrane , cell membrane , chemistry , membrane protein , cell free protein synthesis , protein subunit , atp synthase gamma subunit , chemiosmosis , cell , phospholipid , escherichia coli , biophysics , biology , protein biosynthesis , enzyme , atpase , atp hydrolysis , gene
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.