Structural study of the membrane protein MscL using cell-free expression and solid-state NMR.
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.
Abdine
Alaa
A
UMR 7099, CNRS and Université Paris Diderot, IBPC, 13 rue Pierre et Marie Curie, F-75005 Paris, France.
Verhoeven
Michiel A
MA
Park
Kyu-Ho
KH
Ghazi
Alexandre
A
Guittet
Eric
E
Berrier
Catherine
C
Van Heijenoort
Carine
C
Warschawski
Dror E
DE
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
D029968:Escherichia coli Proteins; D007473:Ion Channels; D008565:Membrane Proteins; C086596:MscL protein, E coli; D011208:Powders