Modulation of the pHLIP transmembrane helix insertion pathway.

The membrane-associated folding/unfolding of pH (low) insertion peptide (pHLIP) provides an opportunity to study how sequence variations influence the kinetics and pathway of peptide insertion into bilayers. Here, we present the results of steady-state and kinetics investigations of several pHLIP variants with different numbers of charged residues, with attached polar cargoes at the peptide's membrane-inserting end, and with three single-Trp variants placed at the beginning, middle, and end of the transmembrane helix. Each pHLIP variant exhibits a pH-dependent interaction with a lipid bilayer. Although the number of protonatable residues at the inserting end does not affect the ultimate formation of helical structure across a membrane, it correlates with the time for peptide insertion, the number of intermediate states on the folding pathway, and the rates of unfolding and exit. The presence of polar cargoes at the peptide's inserting end leads to the appearance of intermediate states on the insertion pathway. Cargo polarity correlates with a decrease of the insertion rate. We conclude that the existence of intermediate states on the folding and unfolding pathways is not mandatory and, in the simple case of a polypeptide with a noncharged and nonpolar inserting end, the folding and unfolding appears as an all-or-none transition. We propose a model for membrane-associated insertion/folding and exit/unfolding and discuss the importance of these observations for the design of new delivery agents for direct translocation of polar therapeutic and diagnostic cargo molecules across cellular membranes.

Parking problem and negative cooperativity of binding of myosin subfragment 1 to F-actin.

Previously we provided evidence that myosin subfragment 1 (S1) can bind either one (state 1) or two actin monomers (state 2) in solution and in muscle fiber. Here we present results of the kinetics study of binding of S1 to F-actin labeled with fluorescent dye pyrene. A transition from state 1 to state 2 depends on probability that the second actin is free, which is high when molar ratio of S1/actin (R) is less than 0.5, and it decreases dramatically when R>2.0 due to the parking problem. The kinetics data obtained at different molar ratios were well fitted by two binding states model. The sequential binding of myosin head initially with one actin monomer and then with the second actin monomer in F-actin can play a key role in force generation by actin-myosin and their directed movement.

An intimate collaboration between peroxisomes and lipid bodies.

Although peroxisomes oxidize lipids, the metabolism of lipid bodies and peroxisomes is thought to be largely uncoupled from one another. In this study, using oleic acid-cultured Saccharomyces cerevisiae as a model system, we provide evidence that lipid bodies and peroxisomes have a close physiological relationship. Peroxisomes adhere stably to lipid bodies, and they can even extend processes into lipid body cores. Biochemical experiments and proteomic analysis of the purified lipid bodies suggest that these processes are limited to enzymes of fatty acid beta oxidation. Peroxisomes that are unable to oxidize fatty acids promote novel structures within lipid bodies ("gnarls"), which may be organized arrays of accumulated free fatty acids. However, gnarls are suppressed, and fatty acids are not accumulated in the absence of peroxisomal membranes. Our results suggest that the extensive physical contact between peroxisomes and lipid bodies promotes the coupling of lipolysis within lipid bodies with peroxisomal fatty acid oxidation.

Energetics of peptide (pHLIP) binding to and folding across a lipid bilayer membrane.

The pH low-insertion peptide (pHLIP) serves as a model system for peptide insertion and folding across a lipid bilayer. It has three general states: (I) soluble in water or (II) bound to the surface of a lipid bilayer as an unstructured monomer, and (III) inserted across the bilayer as a monomeric alpha-helix. We used fluorescence spectroscopy and isothermal titration calorimetry to study the interactions of pHLIP with a palmitoyloleoylphosphatidylcholine (POPC) lipid bilayer and to calculate the transition energies between states. We found that the Gibbs free energy of binding to a POPC surface at low pHLIP concentration (state I-state II transition) at 37 degrees C is approximately -7 kcal/mol near neutral pH and that the free energy of insertion and folding across a lipid bilayer at low pH (state II-state III transition) is nearly -2 kcal/mol. We discuss a number of related thermodynamic parameters from our measurements. Besides its fundamental interest as a model system for the study of membrane protein folding, pHLIP has utility as an agent to target diseased tissues and translocate molecules through the membrane into the cytoplasm of cells in environments with elevated levels of extracellular acidity, as in cancer and inflammation. The results give the amount of energy that might be used to move cargo molecules across a membrane.

Signal transduction in Mimosa pudica: biologically closed electrical circuits.

Biologically closed electrical circuits operate over large distances in biological tissues. The activation of such circuits can lead to various physiological and biophysical responses. Here, we analyse the biologically closed electrical circuits of the sensitive plant Mimosa pudica Linn. using electrostimulation of a petiole or pulvinus by the charged capacitor method, and evaluate the equivalent electrical scheme of electrical signal transduction inside the plant. The discharge of a 100 microF capacitor in the pulvinus resulted in the downward fall of the petiole in a few seconds, if the capacitor was charged beforehand by a 1.5 V power supply. Upon disconnection of the capacitor from Ag/AgCl electrodes, the petiole slowly relaxed to the initial position. The electrical properties of the M. pudica were investigated, and an equivalent electrical circuit was proposed that explains the experimental data.

Mimosa pudica: Electrical and mechanical stimulation of plant movements.

Thigmonastic movements in the sensitive plant Mimosa pudica L., associated with fast responses to environmental stimuli, appear to be regulated through electrical and chemical signal transductions. The thigmonastic responses of M. pudica can be considered in three stages: stimulus perception, electrical signal transmission and induction of mechanical, hydrodynamical and biochemical responses. We investigated the mechanical movements of the pinnae and petioles in M. pudica induced by the electrical stimulation of a pulvinus, petiole, secondary pulvinus or pinna by a low electrical voltage and charge. The threshold value was 1.3-1.5 V of applied voltage and 2 to 10 microC of charge for the closing of the pinnules. Both voltage and electrical charge are responsible for the electro-stimulated closing of a leaf. The mechanism behind closing the leaf in M. pudica is discussed. The hydroelastic curvature mechanism closely describes the kinetics of M. pudica leaf movements.

Biophysics and structure of the patch and the gigaseal.

Interpreting channel behavior in patches requires an understanding of patch structure and dynamics, especially in studies of mechanosensitive channels. High resolution optical studies show that patch formation occurs via blebbing that disrupts normal membrane structure and redistributes in situ components including ion channels. There is a 1-2 microm region of the seal below the patch where proteins are excluded and this may consist of extracted lipids that form the gigaseal. Patch domes often have complex geometries with inhomogeneous stresses due to the membrane-glass adhesion energy (E(a)), cytoskeletal forces, and possible lipid subdomains. The resting tension in the patch dome ranges from 1-4 mN/m, a significant fraction of the lytic tension of a bilayer ( approximately 10 mN/m). Thus, all patch experiments are conducted under substantial, and uneven, resting tension that may alter the kinetics of many channels. E(a) seems dominated by van der Waals attraction overlaid with a normally repulsive Coulombic force. High ionic strength pipette saline increased E(a) and, surprisingly, increased cytoskeletal rigidity in cell-attached patches. Low pH pipette saline also increased E(a) and reduced the seal selectivity for cations, presumably by neutralizing the membrane surface charge. The seal is a negatively charged, cation selective, space with a resistance of approximately 7 gigohm/microm in 100 mM KCl, and the high resistivity of the space may result from the presence of high viscosity glycoproteins. Patches creep up the pipette over time with voltage independent and voltage dependent components. Voltage-independent creep is expected from the capillary attraction of E(a) and the flow of fresh lipids from the cell. Voltage-dependent creep seems to arise from electroosmosis in the seal. Neutralization of negative charges on the seal membrane with low pH decreased the creep rate and reversed the direction of creep at positive pipette potentials.

Adsorption at liquid interfaces: the generalized Langmuir isotherm and interfacial structure.

The thermodynamics of adsorption of amphiphilic surface-active compounds at the interface between two immiscible liquids is considered. At the interface, these molecules are supposed to replace a few of the adsorbed molecules of both solvents. Classical isotherms of adsorption (Frumkin, Langmuir, Henry) were based on the model of nonpenetrable interface, where an adsorbate can substitute only molecules of one solvent. However, at the interface between two immiscible electrolytes, like nonpolar oil-water interfaces, or liquid membrane amphiphilic molecules can substitute molecules of both solvents; therefore, classical isotherms are not applicable in these cases. The generalization of Langmuir and Frumkin isotherms for permeable and nonpermeable interfaces, known as the Markin-Volkov (MV) isotherm, gives the possibility to analyze adsorption and the interfacial structure in a general case. In the present paper, the adsorption isotherms of pentafluorobenzoic acid at the octane-water interface at various pH were measured by the drop-weight method. The thermodynamic parameters of pentafluorobenzoic acid (PFBA) adsorption at the octane-water interface were found. From the measurements of PFBA adsorption, the structure of the octane-water interface was determined. Substitution of one adsorbed octane molecule requires approximately three adsorbed PFBA molecules. This result shows that the orientation of solvent molecules at the interface is different from the bulk solution. Adsorbed octane molecules have a lateral orientation with respect to the interface. Gibbs free energy of adsorption equilibrium and thermodynamic parameters of PFBA adsorption show that the adsorption of PFBA at the octane-water interface is accompanied by a reduction in the attraction between adsorbed PFBA molecules as the pH decreases to the acidic region.

Electrophysiology of pumpkin seeds: Memristors in vivo.

Leon Chua, the discoverer of a memristor, theoretically predicted that voltage gated ion channels can be memristors. We recently found memristors in different plants such as the Venus flytrap, Mimosa pudica, Aloe vera, apple fruits, and in potato tubers. There are no publications in literature about the existence of memristors in seeds. The goal of this work was to discover if pumpkin seeds might have memristors. We selected Cucurbita pepo L., cv. Cinderella, Cucurbita maxima L. cv Warty Goblin, and Cucurbita maxima L., cv. Jarrahdale seeds for this analysis. In these seeds, we found the presence of resistors with memory. The analysis was based on cyclic voltammetry where a memristor should manifest itself as a nonlinear two-terminal electrical element, which exhibits a pinched hysteresis loop on a current-voltage plane for any bipolar cyclic voltage input signal. Dry dormant pumpkin seeds have very high electrical resistance without memristive properties. The electrostimulation by bipolar sinusoidal or triangular periodic waves induces electrical responses in imbibed pumpkin seeds with fingerprints of memristors. Tetraethylammonium chloride, an inhibitor of voltage gated K(+) channels, transforms a memristor to a resistor in pumpkin seeds. NPPB (5-Nitro-2-(3-phenylpropylamino)benzoic acid) inhibits the memristive properties of imbibed pumpkin seeds. The discovery of memristors in pumpkin seeds creates a new direction in the understanding of electrophysiological phenomena in seeds.

Ion fluxes in giant excised cardiac membrane patches detected and quantified with ion-selective microelectrodes.

We have used ion-selective electrodes (ISEs) to quantify ion fluxes across giant membrane patches by measuring and simulating ion gradients on both membrane sides. Experimental conditions are selected with low concentrations of the ions detected on the membrane side being monitored. For detection from the cytoplasmic (bath) side, the patch pipette is oscillated laterally in front of an ISE. For detection on the extracellular (pipette) side, ISEs are fabricated from flexible quartz capillary tubing (tip diameters, 2-3 microns), and an ISE is positioned carefully within the patch pipette with the tip at a controlled distance from the mouth of the patch pipette. Transport activity is then manipulated by solution changes on the cytoplasmic side. Ion fluxes can be quantified by simulating the ion gradients with appropriate diffusion models. For extracellular (intrapatch pipette) recordings, ion diffusion coefficients can be determined from the time courses of concentration changes. The sensitivity and utility of the methods are demonstrated with cardiac membrane patches by measuring (a) potassium fluxes via ion channels, valinomycin, and Na/K pumps; (b) calcium fluxes mediated by Na/Ca exchangers; (c) sodium fluxes mediated by gramicidin and Na/K pumps; and (d) proton fluxes mediated by an unknown electrogenic mechanism. The potassium flux-to-current ratio for the Na/K pump is approximately twice that determined for potassium channels and valinomycin, as expected for a 3Na/2K pump stoichiometery (i.e., 2K/charge moved). For valinomycin-mediated potassium currents and gramicidin-mediated sodium currents, the ion fluxes calculated from diffusion models are typically 10-15% smaller than expected from the membrane currents. As presently implemented, the ISE methods allow reliable detection of calcium and proton fluxes equivalent to monovalent cation currents <1 pA in magnitude, and they allow detection of sodium and potassium fluxes equivalent to <5 pA currents. The capability to monitor ion fluxes, independent of membrane currents, should facilitate studies of both electrogenic and electroneutral ion-coupled transporters in giant patches.

Structure of nonpolarizable water/nitrobenzene interface: potential distribution, ion adsorption, and interfacial tension.

Adsorption of hydrophobic and hydrophilic ions at the nonpolarizable interface between two immiscible electrolyte solutions was investigated. The results were analyzed in three different models: (i) Gouy-Chapman model, (ii) ions as hard spheres, and (iii) ion pair formation at the interface. In the Gouy-Chapman model, an analytical expression for the interfacial tension was obtained. It predicts that interfacial tension should be proportional to the square root of the electrolyte concentration, which does not agree with experimental data. Modeling ions as hard spheres only slightly improves the agreement. The third model of interfacial ion pairing as the main origin of adsorption was analyzed using the amphiphilic isotherm (Markin-Volkov isotherm). A good agreement between ion-pairing theory and experimental values was achieved. The MV isotherm takes into account the limited number of adsorption sites, final size of molecules, complex formation at the interface, and interaction between adsorbed particles. The analysis revealed repulsion between adsorbed tetraalkylammonium ions at the nitrobenzene/water interface and demonstrated linear dependence between adsorption site area and the size of a molecule.

An analytical model of memristors in plants.

The memristor, a resistor with memory, was postulated by Chua in 1971 and the first solid-state memristor was built in 2008. Recently, we found memristors in vivo in plants. Here we propose a simple analytical model of 2 types of memristors that can be found within plants. The electrostimulation of plants by bipolar periodic waves induces electrical responses in the Aloe vera and Mimosa pudica with fingerprints of memristors. Memristive properties of the Aloe vera and Mimosa pudica are linked to the properties of voltage gated K(+) ion channels. The potassium channel blocker TEACl transform plant memristors to conventional resistors. The analytical model of a memristor with a capacitor connected in parallel exhibits different characteristic behavior at low and high frequency of applied voltage, which is the same as experimental data obtained by cyclic voltammetry in vivo.

Morphing structures of the Dionaea muscipula Ellis during the trap opening and closing.

The Venus flytrap is a marvelous plant that has intrigued scientists since the times of Charles Darwin. This carnivorous plant is capable of very fast movements to catch a prey. We found that the maximal speed of the trap closing in the Dionaea muscipula Ellis is about 130,000 times faster than the maximal speed of the trap opening. The mechanism and kinetics of this movement was debated for a long time. Here, the most recent Hydroelastic Curvature Model is applied to the analysis of this movement during closing and opening of the trap with or without a prey. Equations describing the trap movement were derived and verified with experimental data. Chloroform and ether, both anesthetic agents, induce action potentials and close the trap without the mechanical stimulation of trigger hairs. We tested this by dropping 10 µL of ether on the midrib inside the trap without touching any of the mechanosensitive trigger hairs. The trap closed slowly in 10 s. This is at least 20 times slower than the closing of the trap mechanically or electrically. The similar effect can be induced by placing 10 µL of chloroform on the midrib inside the trap, however, the lobes closing time in this case is as fast as closing after mechanical stimulation of the trigger hairs.

Memristors in plants.

We investigated electrical circuitry of the Venus flytrap, Mimosa pudica and Aloe vera. The goal was to discover if these plants might have a new electrical component--a resistor with memory. This element was postulated recently and the researchers were looking for its presence in different systems. The analysis was based on cyclic current-voltage characteristic where the resistor with memory should manifest itself. We found that the electrostimulation of plants by bipolar sinusoidal or triangle periodic waves induces electrical responses in the Venus flytrap, Mimosa pudica and Aloe vera with fingerprints of memristors. Tetraethylammonium chloride, an inhibitor of voltage gated K(+) channels, transforms a memristor to a resistor in plant tissue. Our results demonstrate that a voltage gated K(+) channel in the excitable tissue of plants has properties of a memristor. This study can be a starting point for understanding mechanisms of memory, learning, circadian rhythms, and biological clocks.

Memristors in the electrical network of Aloe vera L.

A memristor is a resistor with memory, which is a non-linear passive two-terminal electrical element relating magnetic flux linkage and electrical charge. Here we found that memristors exist in vivo. The electrostimulation of the Aloe vera by bipolar sinusoidal or triangle periodic waves induce electrical responses with fingerprints of memristors. Uncouplers carbonylcyanide-3-chlorophenylhydrazone and carbonylcyanide-4-trifluoromethoxy-phenyl hydrazone decrease the amplitude of electrical responses at low and high frequencies of bipolar periodic sinusoidal or triangle electrostimulating waves. Memristive behavior of an electrical network in the Aloe vera is linked to the properties of voltage gated ion channels: the K(+) channel blocker TEACl reduces the electric response to a conventional resistor. Our results demonstrate that a voltage gated K(+) channel in the excitable tissue of plants has properties of a memristor. The discovery of memristors in plants creates a new direction in the modeling and understanding of electrical phenomena in plants.

Memristors in the Venus flytrap.

A memristor is a nonlinear element because its current-voltage characteristic is similar to that of a Lissajous pattern for nonlinear systems. We investigated the possible presence of memristors in the electrical circuitry of the Venus flytrap's upper and lower leaves. The electrostimulation of this plant by bipolar sinusoidal or triangle periodic waves induces electrical responses in the upper and lower leaves of the Venus flytrap with fingerprints of memristors. The analysis was based on cyclic voltammetric characteristics where the memristor, a resistor with memory, should manifest itself. Tetraethylammonium chloride, an inhibitor of voltage gated K(+) channels, or NPPB, a blocker of voltage gated Cl(-) and K(+) channels, transform a memristor to a resistor in plant tissue. Uncouplers carbonylcyanide-3-chlorophenylhydrazone (CCCP) and carbonylcyanide-4-trifluoromethoxy-phenyl hydrazone (FCCP) decrease the amplitude of electrical responses at low and high frequencies of bipolar periodic electrostimulating waves. Our results demonstrate that voltage gated K(+) channels in the Venus flytrap have properties of memristors of type 1 and type 2. The discovery of memristors in plants creates a new direction in the modeling and understanding of electrical phenomena in plants.

Memory elements in the electrical network of Mimosa pudica L.

The fourth basic circuit element, a memristor, is a resistor with memory that was postulated by Chua in 1971. Here we found that memristors exist in vivo. The electrostimulation of the Mimosa pudica by bipolar sinusoidal or triangle periodic waves induce electrical responses with fingerprints of memristors. Uncouplers carbonylcyanide-3-chlorophenylhydrazone and carbonylcyanide-4-trifluoromethoxy-phenyl hydrazone decrease the amplitude of electrical responses at low and high frequencies of bipolar sinusoidal or triangle periodic electrostimulating waves. Memristive behavior of an electrical network in the Mimosa pudica is linked to the properties of voltage gated ion channels: the channel blocker TEACl reduces the electric response to a conventional resistor. Our results demonstrate that a voltage gated K(+) channel in the excitable tissue of plants has properties of a memristor. The discovery of memristors in plants creates a new direction in the modeling and understanding of electrical phenomena in plants.

Morphing structures and signal transduction in Mimosa pudica L. induced by localized thermal stress.

Leaf movements in Mimosa pudica, are in response to thermal stress, touch, and light or darkness, appear to be regulated by electrical, hydrodynamical, and chemical signal transduction. The pulvinus of the M. pudica shows elastic properties. We have found that the movements of the petiole, or pinnules, are accompanied by a change of the pulvinus morphing structures. After brief flaming of a pinna, the volume of the lower part of the pulvinus decreases and the volume of the upper part increases due to the redistribution of electrolytes between these parts of the pulvinus; as a result of these changes the petiole falls. During the relaxation of the petiole, the process goes in the opposite direction. Ion and water channel blockers, uncouplers as well as anesthetic agents diethyl ether or chloroform decrease the speed of alert wave propagation along the plant. Brief flaming of a pinna induces bidirectional propagation of electrical signal in pulvini. Transduction of electrical signals along a pulvinus induces generation of an action potential in perpendicular direction between extensor and flexor sides of a pulvinus. Inhibition of signal transduction and mechanical responses in M. pudica by volatile anesthetic agents chloroform or by blockers of voltage gated ion channels shows that the generation and propagation of electrical signals is a primary effect responsible for turgor change and propagation of an excitation. There is an electrical coupling in a pulvinus similar to the electrical synapse in the animal nerves.

Electrotonic and action potentials in the Venus flytrap.

The electrical phenomena and morphing structures in the Venus flytrap have attracted researchers since the nineteenth century. We have observed that mechanical stimulation of trigger hairs on the lobes of the Venus flytrap induces electrotonic potentials in the lower leaf. Electrostimulation of electrical circuits in the Venus flytrap can induce electrotonic potentials propagating along the upper and lower leaves. The instantaneous increase or decrease in voltage of stimulating potential generates a nonlinear electrical response in plant tissues. Any electrostimulation that is not instantaneous, such as sinusoidal or triangular functions, results in linear responses in the form of small electrotonic potentials. The amplitude and sign of electrotonic potentials depend on the polarity and the amplitude of the applied voltage. Electrical stimulation of the lower leaf induces electrical signals, which resemble action potentials, in the trap between the lobes and the midrib. The trap closes if the stimulating voltage is above the threshold level of 4.4V. Electrical responses in the Venus flytrap were analyzed and reproduced in the discrete electrical circuit. The information gained from this study can be used to elucidate the coupling of intracellular and intercellular communications in the form of electrical signals within plants.

Venus flytrap biomechanics: forces in the Dionaea muscipula trap.

Biomechanics of morphing structures in the Venus flytrap has attracted the attention of scientists during the last 140 years. The trap closes in a tenth of a second if a prey touches a trigger hair twice. The driving force of the closing process is most likely due to the elastic curvature energy stored and locked in the leaves, which is caused by a pressure differential between the upper and lower layers of the leaf. The trap strikes, holds and compresses the prey. We have developed new methods for measuring all these forces involved in the hunting cycle. We made precise calibration of the piezoelectric sensor and performed direct measurements of the average impact force of the trap closing using a high speed video camera for the determination of time constants. The new equation for the average impact force was derived. The impact average force between rims of two lobes in the Venus flytrap was found equal to 149 mN and the corresponding pressure between the rims was about 41 kPa. Direct measurements of the constriction force in the trap of Dionaea muscipula was performed during gelatin digestion. This force increases in the process of digestion from zero to 450 mN with maximal constriction pressure created by the lobes reaching to 9 kPa. The insects and different small prey have little chance to escape after the snap of the trap. The prey would need to overpower the "escaping" force which is very strong and can reach up to 4N.