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: 2.85). 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.

Download full-text


Available from: Robin Wray, Sep 27, 2015
12 Reads
    • "Mass spectra of SaMscL C-GFP purified at 4 C in either LDAO or C8E4 (top) prior to incubation at 37 C for 1 hr (bottom). predominantly pentamers in native membranes (Dorwart et al., 2010; Iscla et al., 2011). Interestingly, we found that the interconversion of full-length SaMscL from a pentamer to a tetramer could be stimulated by temperature, in a detergent-dependent manner. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The mechanosensitive channel of large conductance (MscL) acts as an emergency release valve for osmotic shock of bacteria preventing cell lysis. The large pore size, essential for function, requires the formation of oligomers with tetramers, pentamers, or hexamers observed depending on the species and experimental approach. We applied non-denaturing (native) mass spectrometry to five different homologs of MscL to determine the oligomeric state under more than 50 different experimental conditions elucidating lipid binding and subunit stoichiometry. We found equilibrium between pentameric and tetrameric species, which can be altered by detergent, disrupted by binding specific lipids, and perturbed by increasing temperature (37°C). We also established the presence of lipopolysaccharide bound to MscL and other membrane proteins expressed in Escherichia coli, revealing a potential source of heterogeneity. More generally, we highlight the use of mass spectrometry in probing membrane proteins under a variety of detergent-lipid environments relevant to structural biology. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Chemistry & biology 05/2015; 22(5):593-603. DOI:10.1016/j.chembiol.2015.04.016 · 6.65 Impact Factor
  • Source
    • "For MscL, it has not, to our knowledge, been established whether all the MscL channels in a biological membrane are electrophysiologically active, or whether additional nonconducting forms with distinct oligomeric or alternative conformations (such as the precise pairing of TM1 and TM2 between subunits) could be present under certain conditions. While the recent in vivo crosslinking studies of Iscla et al. (2011) "
    [Show abstract] [Hide abstract]
    ABSTRACT: While mechanobiological processes employ diverse mechanisms, at their heart are force-induced perturbations in the structure and dynamics of molecules capable of triggering subsequent events. Among the best characterized force-sensing systems are bacterial mechanosensitive channels. These channels reflect an intimate coupling of protein conformation with the mechanics of the surrounding membrane; the membrane serves as an adaptable sensor that responds to an input of applied force and converts it into an output signal, interpreted for the cell by mechanosensitive channels. The cell can exploit this information in a number of ways: ensuring cellular viability in the presence of osmotic stress and perhaps also serving as a signal transducer for membrane tension or other functions. This review focuses on the bacterial mechanosensitive channels of large (MscL) and small (MscS) conductance and their eukaryotic homologs, with an emphasis on the outstanding issues surrounding the function and mechanism of this fascinating class of molecules.
    Structure 10/2011; 19(10):1356-69. DOI:10.1016/j.str.2011.09.005 · 5.62 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: MscL is a bacterial mechanosensitive channel that protects cells from lysis upon acute decrease in external osmotic environment. It is one of the best characterized mechanosensors known, thus serving as a paradigm of how such molecules sense and respond to stimuli. In addition, the fact that it can be genetically modified, expressed, isolated, and manipulated has led to its proposed use as a triggered nanovalve for various functions including sensors within microelectronic array chips, as well as vesicular-based targeted drug release. X-ray crystallography reveals a homopentameric complex with each subunit containing two transmembrane α-helices (TM1 and TM2) and a single carboxyl terminal α-helix arranging within the complex to form a 5-fold cytoplasmic bundle (CB), whose function and stability remain unclear. In this study, we show three routes that throttle the open channel conductance. When the linker between the TM2 and CB domain is shortened by deletions or constrained by either cross-linking or heavy metal coordination, the conductance of the channel is reduced; in the later two cases, even reversibly. While they have implications for the stability of the CB, these data also provide routes for engineering MscL sensors that are more versatile for potential nanotech devices.
    ACS Nano 12/2011; 6(2):1134-41. DOI:10.1021/nn203703j · 12.88 Impact Factor
Show more