A hexameric transmembrane pore revealed by two-dimensional crystallization of the large mechanosensitive ion channel (MscL) of Escherichia coli.
We have established a reconstitution method of the detergent-solubilized recombinant large mechanosensitive ion channel of Escherichia coli (MscL) that yielded two-dimensional crystals. For that purpose, we have developed a new protocol using Triton X-100 to solubilize and purify the MscL protein. This protocol not only allowed an increase in the protein yield but also made it possible to obtain a homogeneous delipidated and reproducible preparation of the purified protein. When examined by the patch-clamp method MscL channels were found to be fully functional, exhibiting characteristic conductance and activation by pressure. For electron crystallography the homogeneous Triton X-100-purified recombinant MscL was further reconstituted at low lipid-to-protein ratios using Bio-Beads SM2 to remove the detergent. Two-dimensional crystals, exhibiting a p6 plane group symmetry, have been produced and examined by negative stain electron microscopy. Image processing of selected micrographs yielded a projection map at 15-A resolution that provided the first explicit structural information about the molecular boundary and homohexameric organization of the MscL channels in the membrane bilayer.
Saint
N
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Department of Pharmacology, University of Western Australia, Nedlands, WA 6907, Australia.
Lacapère
J J
JJ
Gu
L Q
LQ
Ghazi
A
A
Martinac
B
B
Rigaud
J L
JL
D001426:Bacterial Proteins / Q000737:chemistry; D003460:Crystallization; D003902:Detergents / Q000378:metabolism; D004594:Electrophysiology; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D007473:Ion Channels / Q000737:chemistry* / Q000648:ultrastructure; D008081:Liposomes / Q000378:metabolism; D008854:Microscopy, Electron; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry
D001426:Bacterial Proteins; D003902:Detergents; D029968:Escherichia coli Proteins; D007473:Ion Channels; D008081:Liposomes; C086596:MscL protein, E coli; D011994:Recombinant Proteins