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J Biol Chem, Vol. 273, Issue 24, 14667-14670, June 12, 1998
,,, and
From the 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-Å resolution that
provided the first explicit structural information about the molecular boundary and homohexameric organization of the MscL channels in the
membrane bilayer.
Department of Pharmacology, University of
Western Australia, Nedlands, WA 6907, Australia, § Institut
Curie, Section de Recherche, UMR-CNRS 168 and LRC-CEA 8, 75231 Paris,
France, and ¶ Laboratoire des Biomembranes, ERS CNRS 571, Université Paris-Sud, 91405 Orsay, France
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