Crystal Structure of Escherichia coli MscS, a Voltage-Modulated and Mechanosensitive Channel

The mechanosensitive channel of small conductance (MscS) responds both to stretching of the cell membrane and to membrane depolarization. The crystal structure at 3.9 angstroms resolution demonstrates thatEscherichia coli MscS folds as a membrane-spanning heptamer with a large cytoplasmic region. Each subunit contains three transmembrane helices (TM1, -2, and -3), with the TM3 helices lining the pore, while TM1 and TM2, with membrane-embedded arginines, are likely candidates for the tension and voltage sensors. The transmembrane pore, apparently captured in an open state, connects to a large chamber, formed within the cytoplasmic region, that connects to the cytoplasm through openings that may function as molecular filters. Although MscS is likely to be structurally distinct from other ion channels, similarities in gating mechanisms suggest common structural elements

Breaching the Barrier

Transporter proteins are integral membrane proteins that selectively mediate the passage of molecules across the otherwise impermeable barrier imposed by the phospholipid bilayer that surrounds all cells and organelles. The identification of more than 360 families of transporters through biochemical and genomic analyses highlights the importance of transport processes to cells. Among the most fascinating transporters are those that act as molecular pumps, translocating their substrates across membranes against a concentration gradient; this thermodynamically unfavorable process is powered by coupling to a second, energetically favorable process such as ATP hydrolysis or the movement of a second solute down a transmembrane concentration gradient

Conversion of a Mechanosensitive Channel Protein from a Membrane-Embedded to a Water-Soluble form by Covalent Modification with Amphiphiles

Covalent modification of integral membrane proteins with amphiphiles may provide a general approach to the conversion of membrane proteins into water-soluble forms for biophysical and high-resolution structural studies. To test this approach, we mutated four surface residues of the pentameric Mycobacterium tuberculosis mechanosensitive channel of large conductance (MscL) to cysteine residues as anchors for amphiphile attachment. A series of modified ion channels with four amphiphile groups attached per channel subunit was prepared. One construct showed the highest water solubility to a concentration of up to 4 mg/ml in the absence of detergent. This analog also formed native-like, α-helical homo-pentamers in the absence of detergent as judged by circular dichroism spectroscopy, size-exclusion chromatography and various light-scattering techniques. Proteins with longer, or shorter polymers attached, or proteins modified exclusively with polar cysteine-reactive small molecules, exhibited reduced to no solubility and higher-order aggregation. Electron microscopy revealed a homogeneous population of particles consistent with a pentameric channel. Solubilization of membrane proteins by covalent attachment of amphiphiles results in homogeneous particles that may prove useful for crystallization, solution NMR spectroscopy, and electron microscopy