Mechanosensitive channels open and close in response to applied mechanical stress. The large conductance mechanosensitive channel (MscL) of prokaryotes represent the best characterized member of this family of gated ion channels. We have recently determined the structure of the MscL homolog from Mycobacterium tuberculosis at 3.5 A resolution. This channel consists of a pentamer of identical subunits, organized into transmembrane and extra-membrane domains. Two transmembrane spanning helices are provided by each subunit. At present, MscL provides of the most promising systems for characterizing the gating mechanism of an ion channel in analyses to an open state form of this channel. These structures will define the end points of the gating transition, and in conjunction with the other components of this program project grant, will provide a framework for defining the gating mechanism in this system. Given the similarities in helical packing around the pore observed in MscL and other structurally characterized channels characterized channels, it is anticipated that insights into the gating mechanism of MscL may be of general relevance to other channels. Towards these objectives, we will pursue: 1. Higher resolution crystal structures of closed state conformations for wildtype and selected mutants of MscL. 2. Preparation, crystallization and structure of an open state of MscL, using biochemical, molecular biological and crystallographic methods. 3. Characterization of the solution structure of MscL in detergent micelles and lipid bilayers using site-directed spin-labeling and comparison of these results to the crystal structure. 4. Structural analysis and functional relevance of the extra-membrane domain. 5. Crystallographic analysis of the prokaryotic, methanosensitive YggB channel.
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