title: Structural study of the membrane protein MscL using cell-free expression and solid-state NMR.
abstract: High-resolution structures of membrane proteins have so far been obtained mostly by X-ray crystallography, on samples where the protein is surrounded by detergent. Recent developments of solid-state NMR have opened the way to a new approach for the study of integral membrane proteins inside a membrane. At the same time, the extension of cell-free expression to the production of membrane proteins allows for the production of proteins tailor made for NMR. We present here an in situ solid-state NMR study of a membrane protein selectively labeled through the use of cell-free expression. The sample consists of MscL (mechano-sensitive channel of large conductance), a 75kDa pentameric alpha-helical ion channel from Escherichia coli, reconstituted in a hydrated lipid bilayer. Compared to a uniformly labeled protein sample, the spectral crowding is greatly reduced in the cell-free expressed protein sample. This approach may be a decisive step required for spectral assignment and structure determination of membrane proteins by solid-state NMR.
authors.affiliation: UMR 7099, CNRS and Université Paris Diderot, IBPC, 13 rue Pierre et Marie Curie, F-75005 Paris, France.
authors.forename: Alaa
authors.initials: A
authors.lastname: Abdine
authors.forename: Michiel A
authors.initials: MA
authors.lastname: Verhoeven
authors.forename: Kyu-Ho
authors.initials: KH
authors.lastname: Park
authors.forename: Alexandre
authors.initials: A
authors.lastname: Ghazi
authors.forename: Eric
authors.initials: E
authors.lastname: Guittet
authors.forename: Catherine
authors.initials: C
authors.lastname: Berrier
authors.forename: Carine
authors.initials: C
authors.lastname: Van Heijenoort
authors.forename: Dror E
authors.initials: DE
authors.lastname: Warschawski
mesh_terms: D002474:Cell-Free System / Q000737:chemistry*; D003198:Computer Simulation; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D007473:Ion Channels / Q000737:chemistry* / Q000648:ultrastructure*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008565:Membrane Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D008956:Models, Chemical; D008958:Models, Molecular; D011208:Powders
chemical_list: D029968:Escherichia coli Proteins; D007473:Ion Channels; D008565:Membrane Proteins; C086596:MscL protein, E coli; D011208:Powders