Mechanosensitive channel engineering: A study on the mixing and matching of YnaI and MscS sensor paddles and pores.

Osmotically varying environments are challenging for bacterial cells. Sudden drops in osmolytes cause an increased membrane tension and rupture the cells in the absence of protective mechanisms. One family of protective proteins are mechanosensitive channels of small conductance that open in response to membrane tension.

The MscS-like channel YnaI has a gating mechanism based on flexible pore helices.

The mechanosensitive channel of small conductance (MscS) is the prototype of an evolutionarily diversified large family that fine-tunes osmoregulation but is likely to fulfill additional functions. has six osmoprotective paralogs with different numbers of transmembrane helices. These helices are important for gating and sensing in MscS but the role of the additional helices in the paralogs is not understood.

Structure of the Mechanosensitive Channel MscS Embedded in the Membrane Bilayer.

Since life has emerged, gradients of osmolytes over the cell membrane cause pressure changes in the cell and require tight regulation to prevent cell rupture. The mechanosensitive channel of small conductance (MscS) releases solutes and water when a hypo-osmotic shock raises the pressure in the cell. It is a member of a large family of MscS-like channels found in bacteria, archaea, fungi and plants and model for mechanosensation.

Interaction of the Mechanosensitive Channel, MscS, with the Membrane Bilayer through Lipid Intercalation into Grooves and Pockets.

All membrane proteins have dynamic and intimate relationships with the lipids of the bilayer that may determine their activity. Mechanosensitive channels sense tension through their interaction with the lipids of the membrane. We have proposed a mechanism for the bacterial channel of small conductance, MscS, that envisages variable occupancy of pockets in the channel by lipid chains.

Connecting the Dots between Mechanosensitive Channel Abundance, Osmotic Shock, and Survival at Single-Cell Resolution.

Rapid changes in extracellular osmolarity are one of many insults microbial cells face on a daily basis. To protect against such shocks, and other microbes express several types of transmembrane channels that open and close in response to changes in membrane tension. In , one of the most abundant channels is the mechanosensitive channel of large conductance (MscL).

Bacterial Mechanosensitive Channels.

Mechanosensitive (MS) channels protect bacteria against hypo-osmotic shock and fulfil additional functions. Hypo-osmotic shock leads to high turgor pressure that can cause cell rupture and death. MS channels open under these conditions and release unspecifically solutes and consequently the turgor pressure.

On the mobility, membrane location and functionality of mechanosensitive channels in Escherichia coli.

Bacterial mechanosensitive channels protect cells from structural damage during hypoosmotic shock. MscS, MscL and MscK are the most abundant channels in E. coli and arguably the most important ones in osmoprotection.

The role of lipids in mechanosensation.

The ability of proteins to sense membrane tension is pervasive in biology. A higher-resolution structure of the Escherichia coli small-conductance mechanosensitive channel MscS identifies alkyl chains inside pockets formed by the transmembrane helices (TMs). Purified MscS contains E.

The Structure of YnaI Implies Structural and Mechanistic Conservation in the MscS Family of Mechanosensitive Channels.

Mechanosensitive channels protect bacteria against lysis caused by a sudden drop in osmolarity in their surroundings. Besides the channel of large conductance (MscL) and small conductance (MscS), Escherichia coli has five additional paralogs of MscS that are functional and widespread in the bacterial kingdom. Here, we present the structure of YnaI by cryo-electron microscopy to a resolution of 13 Å.

Properties of the Mechanosensitive Channel MscS Pore Revealed by Tryptophan Scanning Mutagenesis.

Bacterial mechanosensitive channels gate when the transmembrane turgor rises to levels that compromise the structural integrity of the cell wall. Gating creates a transient large diameter pore that allows hydrated solutes to pass from the cytoplasm at rates close to those of diffusion. In the closed conformation, the channel limits transmembrane solute movement, even that of protons.

Probing the structure of the mechanosensitive channel of small conductance in lipid bilayers with pulsed electron-electron double resonance.

Mechanosensitive channel proteins are important safety valves against osmotic shock in bacteria, and are involved in sensing touch and sound waves in higher organisms. The mechanosensitive channel of small conductance (MscS) has been extensively studied. Pulsed electron-electron double resonance (PELDOR or DEER) of detergent-solubilized protein confirms that as seen in the crystal structure, the outer ring of transmembrane helices do not pack against the pore-forming helices, creating an apparent void.

Conformational state of the MscS mechanosensitive channel in solution revealed by pulsed electron-electron double resonance (PELDOR) spectroscopy.

The heptameric mechanosensitive channel of small conductance (MscS) provides a critical function in Escherichia coli where it opens in response to increased bilayer tension. Three approaches have defined different closed and open structures of the channel, resulting in mutually incompatible models of gating. We have attached spin labels to cysteine mutants on key secondary structural elements specifically chosen to discriminate between the competing models.

Characterization of three novel mechanosensitive channel activities in Escherichia coli.

Mechanosensitive channels sense elevated membrane tension that arises from rapid water influx occurring when cells move from high to low osmolarity environments (hypoosmotic shock). These non-specific channels in the cytoplasmic membrane release osmotically-active solutes and ions. The two major mechanosensitive channels in Escherichia coli are MscL and MscS.

Sensing bilayer tension: bacterial mechanosensitive channels and their gating mechanisms.

Mechanosensitive channels sense and respond to changes in bilayer tension. In many respects, this is a unique property: the changes in membrane tension gate the channel, leading to the transient formation of open non-selective pores. Pore diameter is also high for the bacterial channels studied, MscS and MscL.

Tryptophan in the pore of the mechanosensitive channel MscS: assessment of pore conformations by fluorescence spectroscopy.

Structural changes in channel proteins give critical insights required for understanding the gating transitions that underpin function. Tryptophan (Trp) is uniquely sensitive to its environment and can be used as a reporter of conformational changes. Here, we have used site-directed Trp insertion within the pore helices of the small mechanosensitive channel protein, MscS, to monitor conformational transitions.

Physiological analysis of bacterial mechanosensitive channels.

Bacterial mechanosensitive (MS) channels play a significant role in protecting cells against hypoosmotic shock. Bacteria that have been diluted from high osmolarity medium into dilute solution are required to cope with sudden water influx associated with an osmotic imbalance equivalent to 10 to 14 atm. The cell wall is only poorly expansive and the cytoplasmic membrane even less so.

The role of tryptophan residues in the function and stability of the mechanosensitive channel MscS from Escherichia coli.

Tryptophan (Trp) residues play important roles in many proteins. In particular they are enriched in protein surfaces involved in protein docking and are often found in membrane proteins close to the lipid head groups. However, they are usually absent from the membrane domains of mechanosensitive channels.

New dimension in nano-imaging: breaking through the diffraction limit with scanning near-field optical microscopy.

In recent years scanning near-field optical microscopy (SNOM) has developed into a powerful surface analytical technique for observing specimens with lateral resolution equal to or even better than 100 nm. A large number of applications, from material science to biology, have been reported. In this paper, two different kinds of near-field optical microscopy, aperture and scattering-type SNOM, are reviewed together with recent studies in surface analysis and biology.