The mechanoelectrical response of droplet interface bilayer membranes.

Mechanotransduction and interfacial properties in unsupported liquid biomimetic membranes are explored using the droplet-interface bilayer technique. The fluidic monolayer-membrane system afforded by this technique allows for dynamic control over the membrane dimensions and curvature, which under periodic deformations generates capacitive currents (akin to a Kelvin probe), and permits a detailed electrostatic characterization of the boundary layers as well as observation of flexoelectric effects. Both high and low displacement frequency regimes are examined, and the results show that the mechanoelectric signals generated by the membranes may be linked to the membrane electrostatic structure. In addition, we show that periodic membrane bending in a high-frequency regime generates tension sufficient to activate reconstituted mechanosensitive channels.

Hydration properties of mechanosensitive channel pores define the energetics of gating.

Opening of ion channels directly by tension in the surrounding membrane appears to be the most ancient and simple mechanism of gating. Bacterial mechanosensitive channels MscL and MscS are the best-studied tension-gated nanopores, yet the key physical factors that define their gating are still hotly debated. Here we present estimations, simulations and experimental results showing that hydration of the pore might be one of the major parameters defining the thermodynamics and kinetics of mechanosensitive channel gating. We associate closing of channel pores with complete dehydration of the hydrophobic gate (occlusion by 'vapor lock') and formation of two water-vapor interfaces above and below the constriction. The opening path is the expansion of these interfaces, ultimately leading to wetting of the hydrophobic pore, which does not appear to be the exact reverse of the closing path, thus producing hysteresis. We discuss specifically the role of polar groups (glycines) buried in narrow closed conformations but exposed in the open states that change the wetting characteristics of the pore lining and stabilize conductive states of the channels.

Correction of vortex laser beam in a closed-loop adaptive system with bimorph mirror.

The phase correction of a vortex laser beam is undertaken in the closed-loop adaptive system including a Hartmann-Shack wavefront sensor with singular reconstruction technique and a bimorph piezoceramic mirror. After correction the vortex doughnutlike beam is focused into a beam with bright axial spot that considerably increases the Strehl ratio and optical system resolution. Since the phase break cannot be exactly reproduced on the flexible mirror surface, off-axis vortices appear in the far field at the beam periphery.

Wavefront reconstruction of an optical vortex by a Hartmann-Shack sensor.

Reconstruction the phase front of a vortex laser beam is conducted by use of a Hartmann-Shack wavefront sensor. The vortex beam in the form of the Laguerre-Gaussian LG(0)(1) mode is generated with the help of a spiral phase plate. The new reconstruction technique based on measured wavefront gradients allows one to restore the singular phase surface with good accuracy, whereas the conventional least-squares approach fails.

Searching for the molecular arrangement of transmembrane ceramide channels.

Ceramides have been implicated in the initiation of apoptosis by permeabilizing the mitochondrial outer membrane to small proteins, including cytochrome c. In addition, ceramides were shown to form large metastable channels in planar membranes and liposomes, indicating that these lipids permeabilize membranes directly. Here we analyze molecular models of ceramide channels and test their stability in molecular dynamics simulations. The structural units are columns of four to six ceramides H-bonded via amide groups and arranged as staves in either a parallel or antiparallel manner. Two cylindrical assemblies of 14 columns (four or six molecules per column) were embedded in a fully hydrated palmitoyloleoyl-phosphatidylcholine phospholipid bilayer, and simulated for 24 ns in total. After equilibration, the water-filled pore adopted an hourglass-like shape as headgroups of ceramides and phospholipids formed a smooth continuous interface. The structure-stabilizing interactions were both hydrogen bonds between the headgroups (including water-mediated interactions) and packing of the hydrocarbon tails. Ceramide's essential double bond reduced the mobility of the hydrocarbon tails and stabilized their packing. The six-column assembly remained stable throughout a 10-ns simulation. During simulations of four-column assemblies, pairs of columns displayed the tendency of splitting out from the channels, consistent with the previously proposed mechanism of channel disassembly.

200 TW 45 fs laser based on optical parametric chirped pulse amplification.

200 TW peak power has been achieved experimentally using a Cr:forsterite master oscillator at 1250 nm, a stretcher, three optical parametrical amplifiers based on KD*P (DKDP) crystals providing 14.5 J energy in the chirped pulse at 910 nm central wavelength, and a vacuum compressor. The final parametrical amplifier and the compressor are described in detail. Scaling of such architecture to multipetawatt power is discussed.

Structural models of the MscL gating mechanism.

Three-dimensional structural models of the mechanosensitive channel of large conductance, MscL, from the bacteria Mycobacterium tuberculosis and Escherichia coli were developed for closed, intermediate, and open conformations. The modeling began with the crystal structure of M. tuberculosis MscL, a homopentamer with two transmembrane alpha-helices, M1 and M2, per subunit. The first 12 N-terminal residues, not resolved in the crystal structure, were modeled as an amphipathic alpha-helix, called S1. A bundle of five parallel S1 helices are postulated to form a cytoplasmic gate. As membrane tension induces expansion, the tilts of M1 and M2 are postulated to increase as they move away from the axis of the pore. Substantial expansion is postulated to occur before the increased stress in the S1 to M1 linkers pulls the S1 bundle apart. During the opening transition, the S1 helices and C-terminus amphipathic alpha-helices, S3, are postulated to dock parallel to the membrane surface on the perimeter of the complex. The proposed gating mechanism reveals critical spatial relationships between the expandable transmembrane barrel formed by M1 and M2, the gate formed by S1 helices, and "strings" that link S1s to M1s. These models are consistent with numerous experimental results and modeling criteria.

Dipole potentials indicate restructuring of the membrane interface induced by gadolinium and beryllium ions.

The dipole component of the membrane boundary potential, phi(d), is an integral parameter that may report on the conformational state of the lipid headgroups and their hydration. In this work, we describe an experimental approach to measurements of the dipole potential changes, Deltaphi(d), and apply it in studies of Be(2+) and Gd(3+) interactions with membranes composed of phosphatidylserine (PS), phosphatidylcholine (PC), and their mixtures. Deltaphi(d) is determined as the difference between the changes of the total boundary potential, phi(b), measured by the IFC method in planar lipid membranes and the surface potential, phi(s), determined from the electrophoretic mobility of liposomes. The Gouy-Chapman-Stern formalism, combined with the condition of mass balance, well describes the ion equilibria for these high-affinity cations. For the adsorption of Be(2+) and Gd(3+) to PC membranes, and of Mg(2+) to PS membranes, the values of Deltaphi(b) and Deltaphi(s) are the same, indicative of no change of phi(d). Binding of Gd(3+) to PS-containing membranes induces changes of phi(d) of opposite signs depending on the density of ionized PS headgroups in the bilayer. At low density, the induced Deltaphi(d) is negative (-30 mV), consistent with the effect of dehydration of the surface. At maximal density (pure PS, neutral pH), adsorption of Be(2+) or Gd(3+) induces an increase of phi(d) of 35 or 140 mV, respectively. The onset of the strong positive dipole effect on PS membranes with Gd(3+) is observed near the zero charge point and correlates with a six-fold increase of membrane tension. The observed phenomena may reflect concerted reorientation of dipole moments of PS headgroups as a result of ion adsorption and lipid condensation. Their possible implications to in-vivo effects of these high-affinity ions are discussed.

The gating mechanism of the large mechanosensitive channel MscL.

The mechanosensitive channel of large conductance, MscL, is a ubiquitous membrane-embedded valve involved in turgor regulation in bacteria. The crystal structure of MscL from Mycobacterium tuberculosis provides a starting point for analysing molecular mechanisms of tension-dependent channel gating. Here we develop structural models in which a cytoplasmic gate is formed by a bundle of five amino-terminal helices (S1), previously unresolved in the crystal structure. When membrane tension is applied, the transmembrane barrel expands and pulls the gate apart through the S1-M1 linker. We tested these models by substituting cysteines for residues predicted to be near each other only in either the closed or open conformation. Our results demonstrate that S1 segments form the bundle when the channel is closed, and crosslinking between S1 segments prevents opening. S1 segments interact with M2 when the channel is open, and crosslinking of S1 to M2 impedes channel closing. Gating is affected by the length of the S1-M1 linker in a manner consistent with the model, revealing critical spatial relationships between the domains that transmit force from the lipid bilayer to the channel gate.

Stoichiometry of the large conductance bacterial mechanosensitive channel of E. coli. A biochemical study.

MscL, a 15 kDa transmembrane protein, is the only component involved in the formation of a 3 nS channel in the inner membrane of Escherichia coli that opens in response to mechanical or osmotic stress. While previous data had suggested that the functional MscL complex might be a hexamer, a recent crystallographic study of the MscL homologue from M. tuberculosis reveals a pentameric structure. The present work further examines the stoichiometry of the E. coli MscL using a variety of biochemical approaches. Detergent-purified 6His-MscL in solution and MscL in the membrane could be chemically crosslinked with the products displaying ladderlike patterns on SDS gels. Three crosslinking agents (EDC, DMS, and DMA) used at saturating concentrations invariably generated pentamers as the largest product. DSS produced additional bands corresponding to larger complexes although the pentamer band appeared to be the predominant product at high levels of crosslinker. It is not clear whether these extra bands reflect a difference in the crosslinking chemistry of DSS or whether its spacer arm is the longest of those used, or a combination of both facts. For the detergent-solubilized 6His-MscL both sedimentation equilibrium and gel chromatography showed the presence of multiple species. Thus the longer spacer arm could permit both intra- and intercomplex linkages. Nonetheless, the patterns obtained with all agents are consistent with and strongly suggest a pentameric organization for the MscL channel. Expression of MscL as genetically engineered double or triple subunit tandems yields low numbers of functional channels as compared to expressed monomers. The double-tandem assemblies must have an even number of subunits and crosslinking in the membrane confirmed hexamerization. Gel chromatography clearly demonstrated that the channels formed from the double tandems were larger than those formed from WT MscL, consistent with the native channel being pentameric. The observation that both double and triple tandems form channels of normal conductance implies that the pentameric assembly is to some degree independent of the number of subunit repeats in the polypeptide precursor. The channel is thus a pentameric core with the 'extra' subunits left out of the functional complex. From sedimentation equilibrium and size-exclusion chromatography, we also conclude that MscL complexes are not in a dynamic equilibrium with monomers, but are pre-assembled; and thus, their gating properties must result from changes in the conformation of the entire complex induced by the mechanical stress.

Mechanosensitive channels in bacteria as membrane tension reporters.

The purpose of this short review is to discuss recent data on the molecular structure and mechanism of gating of MscL, a mechanosensitive channel of large conductance from Escherichia coli. MscL is the first isolated molecule shown to convert mechanical stress of the membrane into a simple response, the opening of a large aqueous pore. The functional complex appears to be a stable homo-pentamer of 15-kDa subunits, the gating transitions in which are driven by stretch forces conveyed through the lipid bilayer. We have measured the open probability of MscL and the kinetics of transitions as a function of membrane tension. The parameters extracted from the single-channel current recordings and dose-response curves such as the energy difference between the closed, open, and intermediate conducting states, and the transition-related changes in protein dimensions suggest a large conformational rearrangement of the channel complex. The estimations show that in native conditions MscL openings could be driven primarily by forces of osmotic nature. The thermodynamic and spatial parameters reasonably correlate with the available data on the structure of a single MscL subunit and multimeric organization of the complex. Combined with the functional analysis of mutations, these data give grounds to hypotheses on the nature of the channel mechanosensitivity.

Energetic and spatial parameters for gating of the bacterial large conductance mechanosensitive channel, MscL.

MscL is multimeric protein that forms a large conductance mechanosensitive channel in the inner membrane of Escherichia coli. Since MscL is gated by tension transmitted through the lipid bilayer, we have been able to measure its gating parameters as a function of absolute tension. Using purified MscL reconstituted in liposomes, we recorded single channel currents and varied the pressure gradient (P) to vary the tension (T). The tension was calculated from P and the radius of curvature was obtained using video microscopy of the patch. The probability of being open (Po) has a steep sigmoidal dependence on T, with a midpoint (T1/2) of 11.8 dyn/cm. The maximal slope sensitivity of Po/Pc was 0.63 dyn/cm per e-fold. Assuming a Boltzmann distribution, the energy difference between the closed and fully open states in the unstressed membrane was DeltaE = 18.6 kBT. If the mechanosensitivity arises from tension acting on a change of in-plane area (DeltaA), the free energy, TDeltaA, would correspond to DeltaA = 6.5 nm2. MscL is not a binary channel, but has four conducting states and a closed state. Most transition rates are independent of tension, but the rate-limiting step to opening is the transition between the closed state and the lowest conductance substate. This transition thus involves the greatest DeltaA. When summed over all transitions, the in-plane area change from closed to fully open was 6 nm2, agreeing with the value obtained in the two-state analysis. Assuming a cylindrical channel, the dimensions of the (fully open) pore were comparable to DeltaA. Thus, the tension dependence of channel gating is primarily one of increasing the external channel area to accommodate the pore of the smallest conducting state. The higher conducting states appear to involve conformational changes internal to the channel that don't involve changes in area.

Study of protein carbonyls in subcellular fractions isolated from liver and spleen of old and gamma-irradiated rats.

Age- and gamma-irradiation-dependent accumulation of oxidatively modified proteins (measured as carbonyl level) was studied in cytoplasm, mitochondria and nuclei isolated from spleen and liver of 4- and 26-month-old rats. The protein carbonyl levels significantly increased with age in all fractions studied. The carbonyl content was found to be two times higher in the nuclei than in the mitochondria and cytoplasm, which may be related to an extensive modification of lysine and arginine residues in histone molecules. Gamma-Irradiation of rats with 10 Gy caused a rise of protein carbonyls only in their cytoplasm and mitochondria, which was prevented in the animals fed with antioxidants and vitamins for a month before the irradiation. We observed an activation of histone-specific proteases in the nuclei of gamma-irradiated rats. The lack of carbonyl accumulation in the nuclear proteins isolated from tissues of gamma-irradiated animals may be explained by the degradation of oxidized histones by these proteases.

Helicity, membrane incorporation, orientation and thermal stability of the large conductance mechanosensitive ion channel from E. coli.

In this report, we present structural studies on the large conductance mechanosensitive ion channel (MscL) from E. coli in detergent micelles and lipid vesicles. Both transmission Fourier transform infrared spectroscopy and circular dichroism (CD) spectra indicate that the protein is highly helical in detergents as well as liposomes. The secondary structure of the proteins was shown to be highly resistant towards denaturation (25-95 degrees C) based on an ellipticity thermal profile. Amide H+/D+ exchange was shown to be extensive (ca. 66%), implying that two thirds of the protein are water accessible. MscL, reconstituted in oriented lipid bilayers, was shown to possess a net bilayer orientation using dichroic ratios measured by attenuated total-reflection Fourier transform infrared spectroscopy. Here, we present and discuss this initial set of structural data on this new family of ion-channel proteins.

Lipid and cell membranes in the presence of gadolinium and other ions with high affinity to lipids. 2. A dipole component of the boundary potential on membranes with different surface charge.

When Gd3+, a trivalent lanthanide, binds phospholipids with a high affinity, it elicits strong electrostatic effects on the surface of the lipid bilayer. Two experimental methods were applied to monitor the changes in the boundary and surface potentials induced by Gd3+ adsorption on liposomes and planar lipid bilayer membranes (BLM) made from phosphatidylserine (PS), phosphatidylcholine (PC) and their mixtures. The membrane surface charge density was changed by either varying the PS/PC ratio or by changing the degree of PS headgroup ionization in the range of pH between 2.5 and 7.5. The Gouy-Chapman-Stern (GCS) theory combined with the condition of mass balance in the experimental cell was used for quantitative treatment of ion adsorption and related changes in the diffuse part of the electrical double layer (surface potential). Data obtained using microelectrophoresis of liposome suspensions were well described within the framework of the modified GCS theory with constants of 5.10(4) and 10(3) M-1 for Gd3+ association with PS and PC, respectively (Yu. A. Ermakov, A. Z. Averbakh, and S. I. Sukharev, Biol. Membrany 14:434-445 (1997) (in Russian)). The intramembrane field compensation (IFC) technique used to study Gd3+ adsorption on planar lipid bilayers by monitoring the entire boundary potential gave completely different results. An observed drastic difference (approximately 140 mV) between the changes of boundary and surface potential was interpreted as the change in the dipole potential induced by binding of Gd3+. The magnitude of the surface dipole increased with the concentration of PS in PS/PC mixtures and became significant at most negative surface charges (more than 80% of PS in the mixture) and strongly correlated with the degree of PS ionization at different pH. The nature of structural changes at the membrane/water interface induced by Gd(3+)-PS interaction and possible lipid clusterization are discussed in the context of their biological importance.

Role of proteases in activation of apoptosis.

Programmed cell death, or apoptosis, is a physiological cell suicide mechanism, which is triggered in the cells by different stimuli. It has been shown that proteases play a significant role both in the target cell killing by cytotoxic lymphocytes and in the TNF- or anti-Fas-induced cell death. The proteases involved in the early (induction) and late (cell self-destruction) stages of apoptosis are reviewed. It is suggested that the late stages are connected with the activation of a cascade of intracellular proteases, which leads to massive protein destruction. It is likely that the protein destruction is mainly designed for preventing autoimmune response to proteins released from dying cells.

A mechanosensitive channel protein and its gene in E. coli.

Receptor molecules that respond directly to gravity, touch, vibration, or osmotic pressure are inferred from their functions but not yet characterized as isolated proteins or products of cloned genes. These receptors are often in low abundance and in animal and plant tissues that are inaccessible, thus making biochemical analysis difficult. Yet, the application of the sensitive patch-clamp technique to measure transmembrane currents has demonstrated the ubiquity of ion channels whose opening is favored by membrane stretch forces. We have discovered in E. coli the activity of a mechanosensitive ion channel of large conductance (MscL), and have successfully isolated the corresponding protein and gene (Sukharev et al. 1994a). MscL channel appears to respond directly to stretch force in the lipid bilayer since it is active in artificial patches having only highly enriched MscL protein and lipids. Structurally, MscL is an integral membrane protein of only 136 amino-acid residues. Each channel pore is likely to be enclosed by six assembled MscL subunits. Hydropathy analysis suggests that the protein is largely hydrophobic with a more hydrophilic carboxyl tail. Targeted deletions and substitutions show that not all regions of the molecule contribute to channel function; however, strategic single amino-acid changes can alter channel kinetics and mechanosensitivity. MscL and its gene now form the first tangible system to study mechanosensing using a combination of genetic, biochemical, and biophysical techniques.

Neutrophils as a source of putative restriction proteases: degradation of mammalian and yeast proteins monitored by two-dimensional gel electrophoresis.

We have compared the ability of intact neutrophils to degrade a complex substrate of proteins from mammalian and yeast origin. The substrate was obtained by biosynthetic labeling, and subsequent lysis of K562 cells (leukemic cell line) and of yeast culture. The mammalian substrate consisted of 619 and the yeast substrate of 185 different polypeptides, as visualized and represented on two-dimensional gel patterns. Upon incubation of the mammalian substrate with neutrophils, the bulk of spots disappeared so rapidly that after 240 min of incubation only 21 spots were detectable. Just one spot remained unaltered in its intensity throughout the whole period of incubation. About 440 spots reveal a t1/2 shorter than 8 min. Yeast substrate is represented by a smaller number of the starting polypeptides (185) from which 55 spots "survive" the neutrophil treatment. About 30 spots have a t1/2 shorter than 8 min. We conclude that neutrophils are equipped with a potent proteolytic apparatus, and this is capable of eliminating various proteins in a highly efficient manner. The system is much less effective in eliminating proteins from distant species, like yeast. Although the cells governing and regulating the immune system are clearly of lymphoid origin, it might well be that the preimmune task of eliminating self antigens in a manner as predicted in the restriction protease hypothesis is performed by neutrophils.