Conformation of gramicidin A in Triton X‐100 micelles from CD and FTIR data: a clean example of antiparallel double β5.6 helix formation

The linear peptide gramicidin A (gA) forms prototypical ion channels specific for monovalent cations and has been extensively used to study the organization and dynamics of membrane channels. This polymorphic peptide can adopt two different types of structures, the helical dimer β6.3 (‘channel state’) and the double helical structure with two intertwined monomers. The structure of gA in micelles of detergent Triton X‐100 has been studied using CD, Fourier transform infrared, and fluorescence spectroscopy. The results obtained demonstrate that only one thermodynamically stable gA structure, the antiparallel left‐handed double helix β5.6, is formed in this membrane‐mimetic environment. The position of the tryptophan fluorescence maximum at 332 nm is the same as that in phospholipid membranes. The causative factors governing the double helix formation in the micellar medium are discussed on the basis of known physicochemical properties of Triton X‐100. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Biophysical approaches in the study of biomembrane solubilization: quantitative assessment and the role of lateral inhomogeneity

Detergents are amphiphilic molecules widely used to solubilize biological membranes and/or extract their components. Nevertheless, because of the complex composition of biomembranes, their solubilization by detergents has not been systematically studied. In this review, we address the solubilization of erythrocytes, which provide a relatively simple, robust and easy to handle biomembrane, and of biomimetic models, to stress the role of the lipid composition on the solubilization process. First, results of a systematic study on the solubilization of human erythrocyte membranes by different series of non-ionic (Triton, CxEy, Brij, Renex, Tween), anionic (bile salts) and zwitterionic (ASB, CHAPS) detergents are shown. Such quantitative approach allowed us to propose R_e ^sat—the effective detergent/lipid molar ratio in the membrane for the onset of hemolysis as a new parameter to classify the solubilization efficiency of detergents. Second, detergent-resistant membranes (DRMs) obtained as a result of the partial solubilization of erythrocytes by TX-100, C₁₂E₈ and Brij detergents are examined. DRMs were characterized by their cholesterol, sphingolipid and specific proteins content, as well as lipid packing. Finally, lipid bilayers of tuned lipid composition forming liposomes were used to investigate the solubilization process of membranes of different compositions/phases induced by Triton X-100. Optical microscopy of giant unilamellar vesicles revealed that pure phospholipid membranes are fully solubilized, whereas the presence of cholesterol renders the mixture partially or even fully insoluble, depending on the composition. Additionally, Triton X-100 induced phase separation in raft-like mixtures, and selective solubilization of the fluid phase only.     

Estimation of the surface area of a detergent molecule at the interphase boundary of mixed lipid-detergent aggregates

A new approach was proposed for estimating the surface area of a detergent molecule, a _d , at the interphase boundary of mixed lipid-detergent aggregates. The approach is based on consideration of the relationship between geometrical packing parameters of lipid and detergent molecules and the composition of mixed aggregates. The structural transitions between various aggregates are described in terms of the molecular packing parameter, δ, generalized for the case of a two-component lipid-detergent system (Goltsov, A.N. and Barsukov, L.I., J. Biol. Phys., 2000, vol. 26, pp. 27–41). The effective packing parameter, δ_cr, was shown to be a function of the detergent/lipid ratio required for complete solubilization of mixed aggregates, R _e ^sol , the interfacial surface area of a detergent molecule, a _d , and some other molecular packing parameters dependent on the chemical structure of detergent and lipid. The a _d values calculated using the proposed approach are in a fairly good agreement with the experimental data available in literature. The approach ensures effective evaluation of the dependence of a _d on the medium composition, temperature, and the chemical structure of detergent in a broad range of temperatures and detergent/lipid ratios. The conditions of saturation of the lipid bilayer with detergent at the beginning of solubilization were analyzed. The relationship between R _e ^sol and the lipid/detergent ratio required for the saturation of lipid bilayer with detergent, R _e ^sat , was found. A method was developed for R _e ^sat estimation using the R _e ^sol values available in the literature. The results of the present study can be used in the theoretical analysis of mixed lipid-detergent and protein-detergent aggregates.     

Studies on the amphipathic nature of the membrane proteins in Semliki Forest virus

The membrane glycoproteins E₁ and E₂ of Semliki Forest virus form spikes protruding from the external surface of the virion. They have been cleaved off by thermolysin or subtilisin leaving peptide segments in the membrane of the spikeless virus particles with a molecular weight of about 5000 enriched in hydrophobic amino acids. These peptides are soluble in chloroform/methanol and are solubilized into mixed micelles with Triton X100, with sodium dodecyl sulphate and with sodium deoxycholate. Peptide mapping studies show that each membrane glycoprotein has its own lipophilic peptide segment which presumably serves to anchor these proteins to the lipid membrane. The hydrophobic segments of the glycoproteins appear to be shielded from proteolysis not only by the lipids in the intact membrane but also by Triton X100 in the detergent-protein complexes obtained when this detergent is used to remove the lipid and solubilize the proteins.     

Partitioning of Triton X-100, deoxycholate and C10EO8 into bilayers composed of native and hydrogenated egg yolk sphingomyelin

We have used isothermal titration calorimetry (ITC) to study the thermodynamics of Triton X-100 (T_X-100), deoxycholate and decyl octaethylene glycol (C₁₀EO₈) penetration into bilayers composed of native (ESM) and hydrogenated egg yolk sphingomyelin (DHSM). Light scattering measurements were used to study the point of saturation (R_e,sat) and the onset of solubilization of membranes by the detergents. We found that DHSM bilayers at 25 °C were much more resistant to detergent partitioning (lower K) and gave higher reaction enthalpies (ΔH) for all three detergents compared to the ESM bilayer system. Because DHSM lacks double bonds (Δ^4trans and some cis bonds as well), attractive acyl chain interactions are favored in membranes of this lipid class. The high stability and cohesion of DHSM in membranes could be a crucial functional property of this lipid as it is enriched in eye lens membranes.     

Regulation of sodium channel function by bilayer elasticity: the importance of hydrophobic coupling. Effects of Micelle-forming amphiphiles and cholesterol

Membrane proteins are regulated by the lipid bilayer composition. Specific lipid-protein interactions rarely are involved, which suggests that the regulation is due to changes in some general bilayer property (or properties). The hydrophobic coupling between a membrane-spanning protein and the surrounding bilayer means that protein conformational changes may be associated with a reversible, local bilayer deformation. Lipid bilayers are elastic bodies, and the energetic cost of the bilayer deformation contributes to the total energetic cost of the protein conformational change. The energetics and kinetics of the protein conformational changes therefore will be regulated by the bilayer elasticity, which is determined by the lipid composition. This hydrophobic coupling mechanism has been studied extensively in gramicidin channels, where the channel-bilayer hydrophobic interactions link a "conformational" change (the monomer<-->dimer transition) to an elastic bilayer deformation. Gramicidin channels thus are regulated by the lipid bilayer elastic properties (thickness, monolayer equilibrium curvature, and compression and bending moduli). To investigate whether this hydrophobic coupling mechanism could be a general mechanism regulating membrane protein function, we examined whether voltage-dependent skeletal-muscle sodium channels, expressed in HEK293 cells, are regulated by bilayer elasticity, as monitored using gramicidin A (gA) channels. Nonphysiological amphiphiles (beta-octyl-glucoside, Genapol X-100, Triton X-100, and reduced Triton X-100) that make lipid bilayers less "stiff", as measured using gA channels, shift the voltage dependence of sodium channel inactivation toward more hyperpolarized potentials. At low amphiphile concentration, the magnitude of the shift is linearly correlated to the change in gA channel lifetime. Cholesterol-depletion, which also reduces bilayer stiffness, causes a similar shift in sodium channel inactivation. These results provide strong support for the notion that bilayer-protein hydrophobic coupling allows the bilayer elastic properties to regulate membrane protein function.     

Release of remnant plasma membrane from milk fat globules by Triton X-100

The nonionic detergent, Triton X-100, was investigated as an agent for releasing plasma membrane from milk fat globules. The sedimentable material ($\text{50 000 × g}$, 1 h) derived by treating washed goat globules with the detergent (0.2%) was compared to membrane made by the classical globule churning procedure. Characterization included lipid and protein analyses, gel electrophoresis of peptide components, determination of enzymatic activities, and examination with the electron microscope. The results established that the detergent-released material is membrane with similarities to the product by churning. Evaluation of variables revealed that a detergent concentration of 0.1 to 0.2% and reaction temperature of 20–22°C appear optimum with respect to membrane yield when a reaction time of 2 min is employed. At higher detergent concentrations or temperatures removal of phospholipid from the membrane was maximized. Triton X-100 was observed to release membrane from milk fat globules of the goat, human and cow, the latter with a minor procedural modification. The detergent based method is a convenient procedure for obtaining plasma membrane material in good yield for biochemical studies. It also should aid investigations of milk fat globule structure.     

Role of mitochondrial outer membrane in the uncoupling activity of N-terminally glutamate-substituted gramicidin A

Of a series of gramicidin A (gA) derivatives, we have earlier found the peptide [Glu1]gA exhibiting very low toxicity toward mammalian cells, although dissipating mitochondrial membrane potential with almost the same efficiency as gA. Substitution of glutamate for valine at position 1 of the gA amino acid sequence, which is supposed to interfere with the formation of ion-conducting gA channels via head-to-head dimerization, reduces both channel-forming potency of the peptide in planar lipid bilayer membranes and its photonophoric activity in unilamellar liposomes. Here, we compared [Glu1]gA and gA abilities to cause depolarization of the inner mitochondrial membrane in mitochondria and mitoplasts, the latter lacking the outer mitochondrial membrane. Importantly, much less gA was needed to decrease the membrane potential in mitoplasts than in mitochondria, whereas the depolarizing potency of [Glu1]gA was nearly the same in these systems. Moreover, in multilamellar liposomes, [Glu1]gA exhibited more pronounced protonophoric activity than gA, in contrast to the data for unilamellar liposomes. These results allowed us to conclude that [Glu1]gA has a much higher permeability between adjacent lipid membranes than gA. Therefore, the fraction of peptide molecules, reaching the inner mitochondrial membrane upon the addition to cells, is much higher for [Glu1]gA compared to gА. Under these conditions, the decreased cytotoxicity of [Glu1]gA could be associated with its low efficiency as a channel-former dissipating potassium and sodium ion gradients across plasma membrane. The present study highlighted the role of the ability to permeate among various biological membranes for intracellular efficiency of ionophores.     

Comparative study of the interaction of CHAPS and Triton X-100 with the erythrocyte membrane

The zwitterionic detergent CHAPS, a derivative of the bile salts, is widely used in membrane protein solubilization. It is a “facial” detergent, having a hydrophilic side and a hydrophobic back. The objective of this work is to characterize the interaction of CHAPS with a cell membrane. To this aim, erythrocytes were incubated with a wide range of detergent concentrations in order to determine CHAPS partition behavior, and its effects on membrane lipid order, hemolytic effects, and the solubilization of membrane phospholipids and cholesterol. The results were compared with those obtained with the nonionic detergent Triton X-100. It was found that CHAPS has a low affinity for the erythrocyte membrane (partition coefficient K = 0.06 mM^− 1), and at sub-hemolytic concentrations it causes little effect on membrane lipid order. CHAPS hemolysis and phospholipid solubilization are closely correlated. On the other side, binding of Triton X-100 disorders the membrane at all levels, and has independent mechanisms for hemolysis and solubilization. Differential behavior was observed in the solubilization of phospholipids and cholesterol. Thus, the detergent resistant membranes (DRM) obtained with the two detergents will have different composition. The behaviors of the two detergents are related to the differences in their molecular structures, suggesting that CHAPS does not penetrate the lipid bilayer but binds in a flat position on the erythrocyte surface, both in intact and cholesterol depleted erythrocytes. A relevant result for Triton X-100 is that hemolysis is not directly correlated with the solubilization of membrane lipids, as it is usually assumed.     

Membrane damage by a toxin from the sea anemone Stoichactis helianthus. II. Effect of membrane lipid composition in a liposome system

In the first paper of this series, it was shown that a toxin from the sea anemone Stoichactis helianthus increased the permeability of black lipid membranes due to transmembrane channel formation. In the present study, we have used liposomes to examine the reactivity of the toxin with different phospholipids. Membrane damage was assessed by measuring the release of ⁸⁶Rb⁺ and ¹⁴C-labeled membrane lipid. For the different lipids, the rank order of marker release was: sphingomyelin > C18: 2 phosphatidylcholine > C18: 1 phosphatidylcholine > C18: 0 phosphatidylcholine > C16: 0 phosphatidylcholine = C14: 0 phosphatidylcholine. In C14: 0 and C16: 0 phosphatidylcholine liposomes there was no ¹⁴C-labeled lipid release and only 13 to 16% ⁸⁶Rb⁺ release which corresponds to the ⁸⁶Rb⁺ content in the outermost aqueous shell of multilamellar liposomes. This indicates that membrane damage was limited to the outermost bilayer. In liposomes prepared with the other lipids, the extent of release of both markers increased proportionately with the length and the degree of unsaturation of the lipids' acyl side chains. Sphingomyelin liposomes were the most susceptible with 47% of the ¹⁴C-labeled lipid marker and 90% of the ⁸⁶Rb⁺ marker being released. The large extent of ¹⁴C-labeled lipid release is attributed to a detergent-like activity of the toxin which presumably is due to the amphipathic nature of the protein. Thus, the toxin can inflict membrane damage in two ways: (1) channel formation, and (2) detergent action. The importance of one mechanism or the other apparently varies depending on membrane structure and lipid composition.     

Thermodynamics of interaction of octyl glucoside with phosphatidylcholine vesicles: partitioning and solubilization as studied by high sensitivity titration calorimetry

The interaction of the surfactant octyl glucoside (OG) with dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), and soy bean phosphatidylcholine (soy bean PC) was studied using high-sensitivity titration calorimetry. We determined the partition coefficient of OG between water and lipid bilayers and the transfer enthalpy of the surfactant by addition of lipid vesicles to OG monomers or vice versa. Comparison with the micellization enthalpy of the surfactant gives information on differences in the hydrophobic environment of OG in a liquid-crystalline bilayer or a micelle. The average partition coefficient P in mole fraction units for x_e≈0.12–0.2 decreases slightly from 4152 at 27°C to 3479 at 70°C for DMPC and from 4260 to 3879 for soy bean PC, respectively. The transfer enthalpy ΔH^T of OG into lipid vesicles is positive at 27°C and negative at 70°C. Its temperature dependence is larger for the incorporation of OG into DMPC than into soy bean PC vesicles. It is concluded that OG in DMPC vesicles is better shielded from water than in soy bean PC vesicles or in micelles. Titration calorimetry was also used to determine the phase boundaries of the coexistence region of mixed vesicles and mixed micelles in the systems OG/DMPC, OG/DPPC, OG/DSPC, and OG/soy bean PC vesicles at 70°C in the liquid-crystalline phase. DMPC and soy bean PC solubilization was also studied at 27°C to investigate the effect of temperature. The effective surfactant to lipid ratios at saturation, R_e^sat, for all PCs studied are in the range between 1.33–1.72 and the ratios at complete solubilization, R_e^sol, are between 1.79–3.06. At 70°C, the R_e^sat values decrease with increasing chain length of the saturated PC. The ratios depend also slightly on temperature and the degree of unsaturation of the fatty acyl chains. For the OG/soy bean PC system, the coexistence range for mixed vesicles and mixed micelles is larger than for the corresponding PCs with saturated chains.     

Entrapment of soy bean trypsin inhibitor and alpha1-antitrypsin by multilamellar liposomes.

The broad-spectrum protease inhibitors, soy bean trypsin inhibitor and α₁-antitrypsin, were entrapped within anionic multilamellar liposomes; the efficiency of entrapment was 12% for α₁-antitrypsin and 14% for soy bean trypsin inhibitor. Entrapment of ³H-labeled d-glucose, a marker for the aqueous compartment, was dependent upon the nature and concentration of coentrapped antiprotease. Rechromatography of the pooled liposome fractions following solubilization with the detergent Triton X-100 demonstrated latency of the entrapped materials. In addition, entrapment of soy bean trypsin inhibitor, as well as ³H-labeled d-glucose, was shown to be proportional to the net anionie surface charge in the lipid bilayers, suggesting sequestration within the aqueous compartments of the liposomes. This localization was also indicated by the minimal adsorption of antiproteases to anionic liposomes, contrasted with the extensive electrostatic binding of the antiproteases to cationic liposomes.     

Solubilization,purification and reconstitution of Ca-ATPase from bovine pulmonary artery smooth muscle microsomes by different detergents: Preservation of native structure and function of the enzyme by DHPC

The properties of Ca²⁺-ATPase purified and reconstituted from bovine pulmonary artery smooth muscle microsomes {enriched with endoplasmic reticulum (ER)} were studied using the detergents 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C₁₂E₈) and Triton X-100 as the solubilizing agents. Solubilization with DHPC consistently gave higher yields of purified Ca²⁺-ATPase with a greater specific activity than solubilization with C₁₂E₈ or Triton X-100. DHPC was determined to be superior to C₁₂E₈; while that the C₁₂E₈ was determined to be better than Triton X-100 in active enzyme yields and specific activity. DHPC solubilized and purified Ca²⁺-ATPase retained the E1Ca−E1*Ca conformational transition as that observed for native microsomes; whereas the C₁₂E₈ and Triton X-100 solubilized preparations did not fully retain this transition. The coupling of Ca²⁺ transported to ATP hydrolyzed in the DHPC purified enzyme reconstituted in liposomes was similar to that of the native micosomes, whereas that the coupling was much lower for the C₁₂E₈ and Triton X-100 purified enzyme reconstituted in liposomes. The specific activity of Ca²⁺-ATPase reconstituted into dioleoyl-phosphatidylcholine (DOPC) vesicles with DHPC was 2.5-fold and 3-fold greater than that achieved with C₁₂E₈ and Triton X-100, respectively. Addition of the protonophore, FCCP caused a marked increase in Ca²⁺ uptake in the reconstituted proteoliposomes compared with the untreated liposomes. Circular dichroism analysis of the three detergents solubilized and purified enzyme preparations showed that the increased negative ellipticity at 223 nm is well correlated with decreased specific activity. It, therefore, appears that the DHPC purified Ca²⁺-ATPase retained more organized and native secondary conformation compared to C₁₂E₈ and Triton X-100 solubilized and purified preparations. The size distribution of the reconstituted liposomes measured by quasi-elastic light scattering indicated that DHPC preparation has nearly similar size to that of the native microsomal vesicles whereas C₁₂E₈ and Triton X-100 preparations have to some extent smaller size. These studies suggest that the Ca²⁺-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C₁₂E₈ and Triton X-100 in many ways, which is suitable for detailed studies on the mechanism of ion transport and the role of protein–lipid interactions in the function of the membrane-bound enzyme.     

Manipulation of activity and orientation of membrane-reconstituted di-tripeptide transport protein DtpT of Lactococcus lactis

The di-tripeptide transport system (DtpT) of Lactococcus lactis was purified to apparent homogeneity by pre-extraction of crude membrane vesicles with octaethylene glycol monodecyl ether (C₁₀E₈), followed by solubilization with n-dodecyl-beta-D-maltoside (DDM) and chromatography on a Ni-NTA resin. The DtpT protein was reconstituted into detergent-destabilized preformed liposomes prepared from E. coli phospholipid/ phosphatidylcholine. A variety of detergents were tested for their ability to mediate the membrane reconstitution of DtpT and their effectiveness to yield proteoliposomes with a high transport activity. The highest activities were obtained with TX₁₀₀, C₁₂E₈ and DM, whereas DDM yielded relatively poor activities, in particular when this detergent was used at concentrations beyond the onset of solubilization of the preformed liposomes. Parallel with the low activity, significant losses of lipid were observed when the reconstitution was performed at high DDM concentrations. This explained at least part of the reduced transport activity as the DtpT protein was highly dependent on the final lipid-to-protein ratios in the proteoliposomes. Consistent with the difference in mechanism of DDM- and TX₁₀₀-mediated membrane protein reconstitution, the orientation of the DtpT protein in the membrane was random with DDM and inside-in when T₁₀₀ was used. The methodology to determine the orientation of membrane-reconstituted proteins from the accessibility of cysteines for thiol-specific reagents is critically evaluated.     

The β-glucoside system of Escherichia coli III. Properties of A P–HPr: β-Glucoside phosphotransferase extracted from membranes with detergent

A P-HPr:β-glucoside phosphotransferase (enzyme II^bgl)

1 The nomenclautre of the enzymes II is that suggested by Lin (1)

has been extracted from membranes of a β-glucoside fermenting strain of Escherichia coli K 12 using the nonionic detergent Triton X–100. The extracted enzyme was rendered virtually free of both lipid and detergent by chromatography on DEAE-cellulose. At this stage, the partially purified enzyme had negligible activity, but activity was restored effectively by the addition of (1) nonionic detergents of the Tween or Triton series and (2) crude E. coli phospholipids or an anionic lipid enriched fraction, but not phosphatidylethanolamine. Detergent activators were most effective at or near the critical micelle concentration, but were inhibitory when added at concentrations above the critical micelle concentration. In order to obtain maximal initial rates of phosphotransferase activity, it was necessary to incubate the extracted, partially purified enzyme with detergent activator and HPr prior to the addition of the other assay system components. High detergent concentration inhibited the initial rate of phosphorylation by interfering with an essential step (or steps) that occur during this preliminary incubation. The activation occuring during the preliminary incubation was also highly temperature dependent; a precipitous decrease in activation was detected below 16° when Tween 40 was employed as the detergent activator. Phosphorylation mediated by the membrane associated form of the phosphotransferase was not influenced by the physical state of the lipid components of the membrane. This is in marked contrast to the properties of the phosphorylation reaction mediated by the phosphotransferase in intact cells.     

Solubilization of pituitary receptors for thyrotropin-releasing hormone

Receptors for thyrotropin-releasing hormone were solubilized by Triton X-100. Membrane fractions from GH₃ pituitary tumor cells were incubated with thyrotropin-releasing hormone in order to saturate specific receptor sites before the addition of detergent. The amount of protein-bound hormone solubilized by Triton X-100 was proportional to the fractional saturation of specific membrane receptors. Increasing detergent: protein ratios from 0.5 to 20 led to a progressive loss of hormone · receptor complex from membrane fractions with a concomitant increase in soluble protein-bound hormone. The soluble hormone · receptor complex was not retained by 0.22 μm filters and remained soluble after ultracentrifugation. Following incubation with high (2.5–10%) concentration of Triton X-100 and other non-ionic detergents, or following repeated detergent extraction, at least 18% of specifically bound thyrotropin-releasing hormone remained associated with particulate material. Unlike the hormone receptor complex, the free hormone receptor was inactivated by Triton X-100. A 50% loss of binding activity was obtained with 0.01% Triton X-100, corresponding to a detergent: protein ratio of 0.033.The hormone · receptor complex was included in Sepharose 6B and exhibited an apparent Stokes radius of 46 Å in buffers containing Triton X-100. The complex aggregated in detergent-free buffers. Soluble hormone receptors were separated from excess detergent and thyrotropin-releasing hormone by chromatography on DEAE-cellulose. Thyrotropin-releasing hormone dissociated from soluble receptors with a half-time of 120 min at 0°c, while the membrane hormone · receptor complex was stable for up to 5 h at 0°C.     

Characterization of Horizontal Lipid Bilayers as a Model System to Study Lipid Phase Separation

Artificial lipid membranes are widely used as a model system to study single ion channel activity using electrophysiological techniques. In this study, we characterize the properties of the artificial bilayer system with respect to its dynamics of lipid phase separation using single-molecule fluorescence fluctuation and electrophysiological techniques. We determined the rotational motions of fluorescently labeled lipids on the nanosecond timescale using confocal time-resolved anisotropy to probe the microscopic viscosity of the membrane. Simultaneously, long-range mobility was investigated by the lateral diffusion of the lipids using fluorescence correlation spectroscopy. Depending on the solvent used for membrane preparation, lateral diffusion coefficients in the range D_lat = 10-25 μm²/s and rotational diffusion coefficients ranging from D_rot = 2.8 − 1.4 × 10⁷ s⁻¹ were measured in pure liquid-disordered (L_d) membranes. In ternary mixtures containing saturated and unsaturated phospholipids and cholesterol, liquid-ordered (L_o) domains segregated from the L_d phase at 23°C. The lateral mobility of lipids in L_o domains was around eightfold lower compared to those in the L_d phase, whereas the rotational mobility decreased by a factor of 1.5. Burst-integrated steady-state anisotropy histograms, as well as anisotropy imaging, were used to visualize the rotational mobility of lipid probes in phase-separated bilayers. These experiments and fluorescence correlation spectroscopy measurements at different focal diameters indicated a heterogeneous microenvironment in the L_o phase. Finally, we demonstrate the potential of the optoelectro setup to study the influence of lipid domains on the electrophysiological properties of ion channels. We found that the electrophysiological activity of gramicidin A (gA), a well-characterized ion-channel-forming peptide, was related to lipid-domain partitioning. During liquid-liquid phase separation, gA was largely excluded from L_o domains. Simultaneously, the number of electrically active gA dimers increased due to the increased surface density of gA in the L_d phase.     

Analyses of Monascus pigment secretion and cellular morphology in non‐ionic surfactant micelle aqueous solution

Monascus pigments produced by Monascus spp. are widely used as natural food colourants. Extractive fermentation technology can facilitate the secretion of intracellular Monascus pigments into extracellular non‐ionic surfactant micelle aqueous solution, so as to avoid the feedback inhibition and decomposition. In this study, behaviour of the trans‐membrane secretion of Monascus pigments was investigated using morphological and spectroscopic analyses. Laser scanning confocal microscopy (LSCM) traced that pigment secretion occurred through rapid trans‐membrane permeation in 4 min, with a simultaneous conversion in pigment characteristics. Approximately 50% of intracellular pigments (AU₄₇₀) extracted to extracellular broth with 40 g l^?1 Triton X‐100, indicating the capacity for pigment extraction was limited by the saturation concentrations of surfactant. Scanning electron microscope (SEM) and transmission electron microscope (TEM) imaging showed some damage in the cell wall but an intact cell membrane with a slightly increased mycelial diameter. However, the physiological properties of the cell membrane, including integrity, fluorescence intensity and permeability, were altered. A diagram was provided to demonstrate the behaviour of Monascus pigment secretion induced by Triton X‐100. This study lays a foundation for the further investigation of Monascus pigment metabolism and secretion in extractive fermentation.     

Factors determining detergent resistance of erythrocyte membranes

The degree of detergent insolubility of cell membranes is a useful parameter to test the strength of lipid–lipid interactions relative to lipid–detergent interactions. Thus, solubility studies could give insights about lipid–lipid interactions relevant in domain formation. In this work we perform a detailed study of the solubilization of four different erythrocyte membrane systems: intact human and bovine erythrocytes, and human and bovine erythrocytes depleted in cholesterol with methyl-β-cyclodextrin. Each system was incubated with different concentrations of the non-ionic detergent Triton X-100, and the insoluble fraction was characterized by determining cholesterol and phosphorus content. A distinct solubilization behavior was obtained for the four systems, which was quantified by a “detergent resistance parameter” obtained from the fit of the solubility curves. In order to correlate these findings with membrane structural parameters, we quantify the degree of acyl chain order/rigidity of the original membranes by EPR spectroscopy, finding that detergent resistance is higher when acyl chains are more rigid. Regarding compositional properties, we found a good correlation between detergent resistance parameters and the total amount of cholesterol plus sphingomyelin in the original membranes. Our results suggest that a high degree of acyl chain packing is the determinant membrane factor for resistance to the action of Triton X-100 in erythrocytes.     

Effect of Linker Length on Avidin Binding to Biotinylated Gramicidin A

Biotinylated gramicidins are an important component of the AMBRI^® “ion channel switch™” biosensor. These gramicidin A (gA) analogues have a biotin attached to the C-terminus of gA via a number of aminocaproyl linker groups (X). The structure of gA5XB has been determined in deuterated sodium dodecyl sulfate micelles and is similar to native gA and other modified gA analogues. The biotin and aminocaproyl groups were mobile and located in the aqueous phase and when avidin was added, NMR and MS studies showed that gA5XB bound more effectively to avidin than gA2XB. The length and flexibility of the linker appears to be important for biotin–avidin binding and, in the AMBRI^® biosensor, gA5XB is a more effective gated ion channel than gA2XB. The conformation and dynamics of the aminocaproyl linker groups were investigated using ²H solid-state NMR. Deuterated aminocaproyl linkers were coupled to gA and incorporated into oriented bilayers in order to analyse the order and dynamics of the aminocaproyl linker. The small ²H splittings and the T ₁ relaxation times indicated that the aminocaproyl linker is undergoing fast rotation in phospholipid bilayers. Native d ₄ -gA as well as d ₄ -gA2XB, where the ethanolamine has been deuterated, were also incorporated into oriented bilayers. Solid-state ²H NMR data showed that the addition of the linker group restricted the mobility of the ethanolamine. However, these modifications to the C-terminus of gA did not interfere with ion channel function and clarify how the biotinylated gA analogues perform in the lipid bilayer as part of the AMBRI^® biosensor.Australian Peptide Conference Issue.