The oligomeric state of the truncated mechanosensitive channel of large conductance shows no variance in vivo.

Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas, USA.
Protein Science (Impact Factor: 2.86). 09/2011; 20(9):1638-42. DOI: 10.1002/pro.686
Source: PubMed

ABSTRACT The mechanosensitive channel of large conductance (MscL) from E. coli serves as an emergency release valve allowing the cell to survive acute osmotic downshock. It is one of the best studied mechanosensitive channels and serves as a paradigm for how a protein can sense and respond to membrane tension. Two MscL crystal structures of the orthologs M. tuberculosis and S. aureus have been solved showing pentameric and tetrameric structures, respectively. Several studies followed to understand whether the discrepancy in their stoichiometry was a species difference or a consequence of the protein manipulation for crystallization. Two independent studies now agree that the full-length S. aureus MscL is actually a pentamer, not tetramer. While detergents appear to play a role in modifying the oligomeric state of the protein, a cytoplasmic helical bundle has also been implicated. Here, we evaluate the role of the C-terminal region of S. aureus MscL in the oligomerization of the channel in native membranes by using an in vivo disulfide-trapping technique. We find that the oligomeric state of S. aureus MscLs with different C-terminal truncations, including the one used to obtain the tetrameric S. aureus MscL crystal structure, are pentamers in vivo. Thus, the C-terminal domain of the S. aureus protein only plays a critical role in the oligomeric state of the SaMscL protein when it is solubilized in detergent.

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    ABSTRACT: Mechanosensitive channels are integral components for the response of bacteria to osmotic shock. The mechanosensitive channel of large conductance (MscL) responds to extreme turgor pressure increase that would otherwise lyse the cellular membrane. MscL has been studied as a model mechanosensitive channel using both structural and functional approaches. We will summarize the structural data and discuss outstanding questions surrounding the gating mechanism of this homo-oligomeric channel that has ~3 nS conductance. Specifically, we will explore the following: (1) the variability in oligomeric state that has been observed, (2) the open pore size measurements, and (3) the role of the C-terminal coiled coil domain for channel function. The oligomeric state of MscL has been characterized using various techniques, with a pentamer being the predominant form; however, the presence of mixtures of oligomers in the membrane is still uncertain. In the absence of structural data for the open state of MscL, the diameter of the open state pore has been estimated by several different approaches, leading to a current estimate between 25 and 30 Å. While the C-terminal domain is highly conserved among MscL homologues, it is not required for activity in vivo or in vitro. This domain is likely to remain intact during the gating transition and perform a filtering function that retains valuable osmolytes in the cytosol. Overall, studies of MscL have provided significant insight to the field, and serve as a paradigm for the analysis of non-homologous, eukaryotic mechanosensitive channel proteins.
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    ABSTRACT: The crystal structure of the cytoplasmic domain (CTD) from the mechanosensitive channel of large conductance (MscL) in E. coli has been determined at 1.45 Å resolution. This domain forms a pentameric coiled coil similar to that observed in the structure of MscL from M. tuberculosis and also found in the Cartilage Oligomeric Matrix Protein (COMPcc). It contains canonical hydrophobic and atypical ionic interactions compared to previously characterized coiled coil structures. Thermodynamic analysis indicates that while the free EcMscL-CTD is less stable than other coiled coils, it is likely to remain folded in context of the full-length channel.
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    ABSTRACT: MscL, the highly conserved bacterial mechanosensitive channel of large conductance, is one of the best studied mechanosensors. It is a homopentameric channel that serves as a biological emergency release valve that prevents cell lysis from acute osmotic stress. We previously showed that the periplasmic region of the protein, particularly a single residue located at the TM1/periplasmic loop interface, F47 of Staphylococcus aureus and I49 of Escherichia coli MscL, plays a major role in both the open dwell time and mechanosensitivity of the channel. Here, we introduced cysteine mutations at these sites and found they formed disulfide bridges that decreased the channel open dwell time. By scanning a likely interacting domain, we also found that these sites could be disulfide trapped by addition of cysteine mutations in other locations within the periplasmic loop of MscL, and this also led to rapid channel kinetics. Together, the data suggest structural rearrangements and protein-protein interactions that occur within this region upon normal gating, and further suggest that locking portions of the channel into a transition state decreases the stability of the open state.
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