PMID:9614061
| Citation |
Saint, N, Lacapère, JJ, Gu, LQ, Ghazi, A, Martinac, B and Rigaud, JL (1998) A hexameric transmembrane pore revealed by two-dimensional crystallization of the large mechanosensitive ion channel (MscL) of Escherichia coli. J. Biol. Chem. 273:14667-70 |
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| Abstract |
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. |
| Links | |
| Keywords |
Bacterial Proteins/chemistry; Crystallization; Detergents/metabolism; Electrophysiology; Escherichia coli/chemistry; Escherichia coli Proteins; Ion Channels/chemistry; Ion Channels/ultrastructure; Liposomes/metabolism; Microscopy, Electron; Patch-Clamp Techniques; Protein Conformation; Recombinant Proteins/chemistry |
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