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BBA - Biomembranes (v.1828, #4)

Editorial Board (pp. i).

K⁺-induced conformational changes in the trimeric betaine transporter BetP monitored by ATR-FTIR spectroscopy by Filiz Korkmaz; Susanne Ressl; Christine Ziegler; Mantele Werner Mäntele (pp. 1181-1191).

The trimeric Na⁺-coupled betaine symporter BetP from Corynebactrium glutamicum adjusts transport activity according to the external osmolality. BetP senses the increasing internal K⁺ concentration, which is an immediate consequence of osmotic upshift in C. glutamicum. It is assumed that BetP specifically binds potassium to yet unidentified binding sites, thereby inducing conformational changes resulting in activation. Atomic structures of BetP were obtained in the absence of potassium allowing only a speculative glimpse on a putative mechanism of K⁺-induced transport activation. The structural data suggest that activation in BetP is crucially linked to its trimeric state involving an interaction network between several arginines and glutamates and aspartates. Here, we describe the effect of K⁺-induced activation on the specific ionic interaction sites in terminal domains and loops and on the protomer–protomer interactions within the trimer studied by ATR-FTIR spectroscopy. We suggest that arginine and aspartate and/or glutamate residues at the trimeric interface rearrange upon K⁺-induced activation, although they remain assembled in an interaction network. Our data propose a two-step mechanism comprising first a change in solvent exposure of charged residues and second a modification of their interaction sites in a partner-switching manner. FTIR reveals a higher α-helical content than expected from the X-ray structures that we attribute to the structurally unresolved N-terminal domain modulating regulation. In situ¹H/²H exchange studies point toward an altered exposure of backbone regions to buffer solution upon activation, most likely due to conformational changes in both terminal domains, which further affects ionic interactions within the trimer.Display Omitted► N-terminus of the protein is suggested to have partial helical structure. ► BetP under activating conditions is more exposed to buffer than its inactive state. ► BetP activation induces salt bridge formation between Arg and Asp/Glu. ► Regulatory interaction networks between protomers are maintained during activation. ► Interacting residues of adjacent protomers switch partner during activation.

Keywords: Abbreviations; TMH; transmembrane helix; ATR-FTIR; attenuated total reflection Fourier transform infrared spectroscopy; LB; lysogeny broth; DDM; β-Dodecyl-maltoside; KPi; potassium phosphate; RMS; root mean square; EPR; electron paramagnetic resonance; AFM; atomic force miscroscopy; a.a.; amino acidSecondary transporter; BetP; Transport regulation; Side chain subtraction; Secondary structure; Curve fitting

Bubble nucleation in lipid bilayers: A mechanism for low frequency ultrasound disruption by Steven P. Wrenn; Eleanor Small; Nily Dan (pp. 1192-1197).

Recent experiments have shown that low frequency ultrasound (LFUS) induces leakage from lipid vesicles. However, the mechanism by which LFUS disrupts the lipid bilayer structure is not clear. In this paper we develop a theoretical model to test the possibility that gas molecule partitioning from the aqueous media into the lipid bilayer core can lead to the nucleation of microscale gas bubbles. If those can, indeed, form, then their presence in the lipid bilayer and interactions with an ultrasound field can cause bilayer disruption and leakage. The model derived here for the nucleation of stable bubbles accounts for the ‘surface tension’ that the lipid bilayer exerts on the bubble, a result of the associated disruption of the lipid packing. The model predicts that the probability of bubble nucleation is highly sensitive to the bilayer thickness, and largely insensitive to the bilayer phase. The probability of stable bubble formation is shown to correlate with experimentally measured sensitivity of lipid bilayers to LFUS, suggesting that membrane disruption may be due to embedded bubbles that nucleated in the bilayer.Display Omitted► Gas molecules partition into lipid bilayers, where they can form micro bubbles. ► Bubble formation is controlled by a surface tension arising from lipid deformation. ► Bubble formation probability is found to be highly sensitive to bilayer thickness. ► Bubble formation probability correlates with measured US-induced leakage.

Keywords: Bubble nucleation; Lipid deformation; Low frequency ultrasound

Modeling the endosomal escape of cell-penetrating peptides using a transmembrane pH gradient by Fatemeh Madani; Rania Abdo; Staffan Lindberg; Hisaaki Hirose; Shiroh Futaki; Ülo Langel; Graslund Astrid Gräslund (pp. 1198-1204).

Cell-penetrating peptides (CPPs) can internalize into cells with covalently or non-covalently bound biologically active cargo molecules, which by themselves are not able to pass the cell membrane. Direct penetration and endocytosis are two main pathways suggested for the cellular uptake of CPPs. Cargo molecules which have entered the cell via an endocytotic pathway must be released from the endosome before degradation by enzymatic processes and endosomal acidification. Endosomal entrapment seems to be a major limitation in delivery of these molecules into the cytoplasm. Bacteriorhodopsin (BR) asymmetrically introduced into large unilamellar vesicles (LUVs) was used to induce a pH gradient across the lipid bilayer. By measuring pH outside the LUVs, we observed light-induced proton pumping mediated by BR from the outside to the inside of the LUVs, creating an acidic pH inside the LUVs, similar to the late endosomes in vivo. Here we studied the background mechanism(s) of endosomal escape. 20% negatively charged LUVs were used as model endosomes with incorporated BR into the membrane and fluorescein-labeled CPPs entrapped inside the LUVs, together with a fluorescence quencher. The translocation of different CPPs in the presence of a pH gradient across the membrane was studied. The results show that the light-induced pH gradient induced by BR facilitates vesicle membrane translocation, particularly for the intermediately hydrophobic CPPs, and much less for hydrophilic CPPs. The presence of chloroquine inside the LUVs or addition of pyrenebutyrate outside the LUVs destabilizes the vesicle membrane, resulting in significant changes of the pH gradient across the membrane.pH-induced peptide membrane translocation.Display Omitted► The background mechanism(s) of endosomal escape are studied. ► Large unilamellar vesicles with membrane inserted bacteriorhodopsin were used. ► Fluorescein-labeled cell-penetrating peptides were encapsulated inside the vesicles. ► Light-induced pH gradient by bacteriorhodopsin caused peptide membrane translocation. ► The response to the pH gradient was stronger for more hydrophobic peptides.

Keywords: Abbreviations; CPP; cell-penetrating peptide; BR; Bacteriorhodopsin; Pyrenebutyrate; 4-(1-pyrenyl)-butyric acid; CQ; chloroquine; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3[phospho-rac-(1-glycerol)]; BMP; sn; -(3-oleoyl-2-hydroxy)-glycerol-1-phospho-; sn; -1′-(3′-oleoyl-2′-hydroxy)-glycerol (ammonium salt); LUV; large unilamellar vesicle; R9; oligoarginine nonamer; TP10; transportan 10; pVEC; vascular endothelial cadherin peptide; TAT; transactivator of transcription peptide; ON; oligonucleotide; OG; n-octyl-β-D-glucopyranoside; FITC; fluorescein isothiocyanate; DLS; dynamic light scatteringCell-penetrating peptide; Endosomal escape; Bacteriorhodopsin; Large unilamellar vesicle; Fluorescein-label; Membrane translocation

Surface pressure induced structural transitions of an amphiphilic peptide in pulmonary surfactant systems by an in situ PM-IRRAS study by Hiromichi Nakahara; Sannamu Lee; Osamu Shibata (pp. 1205-1213).

Pulmonary surfactant model peptide, Hel 13–5, in binary and ternary lipid mixtures has been characterized employing the polarization–modulation infrared reflection–absorption spectroscopy (PM-IRRAS) in situ at the air–water interface for a monolayer state and the polarized ATR-FTIR for a bilayer film. In the bilayer form, Hel 13–5 predominantly adopts an α-helical secondary structure in the lipid mixtures. It had been made clear from CD measurements that the Hel 13–5 structure is mainly in the α-helical form in aqueous solutions. In the monolayer state, however, the secondary structure of Hel 13–5 exhibits an interconversion of the α-helix into β-sheet with increasing surface pressures. The difference in the secondary structure is attributed to formation of a surface-associated reservoir just below the surface monolayer. The reservoir formation is a key function of pulmonary surfactants and is induced by a squeeze-out of the fluid components in their monolayers. Compression and expansion cycles of the monolayers generate a hysteresis in molecular orientation of the lipid monolayer as well as in peptide structure. The formation and deformation of reservoirs are, in common, deeply related to the hysteresis behavior. Thus, the transition of peptide structures across the interface is a quite important matter to clarify the role and its mechanism of the reservoirs in pulmonary functions. The present study primarily reveals roles of the anionic lipids in control of the peptide secondary structure. Accordingly, it is demonstrated that they prevent the protein structure transition from α-helix into β-sheet by incorporating the peptide during the squeeze-out event.Display Omitted► Peptide structure transition from α-helix to β-sheet takes place during compression. ► Formation of surface-associated reservoirs works to preserve α-helix of the peptide. ► Release of squeezed-out molecules is accelerated by partially charged PA. ► PG assumes strong resistance to the peptide structure transition during compression.

Keywords: RDS; Langmuir monolayer; Lung surfactant; DPPC; PG; SP-B

Molecular basis of the facilitation of the heterooligomeric GIRK1/GIRK4 complex by cAMP dependent protein kinase by Fritz Treiber; Christian Rosker; Tal Keren-Raifman; Bibiane Steinecker; Astrid Gorischek; Nathan Dascal; Wolfgang Schreibmayer (pp. 1214-1221).

G-protein activated inwardly rectifying K⁺ channels (GIRKs) of the heterotetrameric GIRK1/GIRK4 composition mediate IK+_ACh in atrium and are regulated by cAMP dependent protein kinase (PKA). Phosphorylation of GIRK1/GIRK4 complexes promotes the activation of the channel by the G-protein Gβγ-dimer (“heterologous facilitation”). Previously we reported that 3 serines/threonines (S/Ts) within the GIRK1 subunit are phosphorylated by the catalytic subunit of PKA (PKA-cs) in-vitro and are responsible for the acute functional effects exerted by PKA on the homooligomeric GIRK1^F137S (GIRK1^⁎) channel. Here we report that homooligomeric GIRK4^WT and GIRK4^S143T (GIRK4^⁎) channels are clearly regulated by PKA phosphorylation. Heterooligomeric channels of the GIRK1^{S385CS401CT407C}/GIRK4^WT composition, where the GIRK1 subunit is devoid of PKA mediated phosphorylation, exhibited reduced but still significant acute effects (reduction during agonist application was ≈49% compared to GIRK1^WT/GIRK4^WT). Site directed mutagenesis of truncated cytosolic regions of GIRK4 revealed four serines/threonines (S/Ts) that were heavily phosphorylated by PKA-cs in vitro. Two of them were found to be responsible for the acute effects exerted by PKA in vivo, since the effect of cAMP injection was reduced by ≈99% in homooligomeric GIRK4^{⁎T199CS412C} channels. Coexpression of GIRK1^WT/GIRK4^T199CS412C reduced the acute effect by ≈65%. Only channels of the GIRK1^{S385CS401CT407C}/GIRK4^T199CS412C composition were practically devoid of PKA mediated effects (reduction by ≈97%), indicating that both subunits contribute to the heterologous facilitation of IK+_ACh.Display Omitted► Phosphorylation of GIRKs is the prerequisite for activation by G-proteins (=heterologous facilitation). ► Direct phosphorylation by PKA was found to be responsible for GIRK4 regulation. ► GIRK4 subunits contribute substantially to regulation of heterooligomeric GIRK1/GIRK4 channels by PKA in vivo. ► Mechanistic insight how G-protein/effector interaction is modulated by effector phosphorylation is provided.

Keywords: GIRK1; GIRK4; PKA; cAMP; Heterologous facilitation

Modified lipid and protein dynamics in nanodiscs by Mors Karsten Mörs; Christian Roos; Frank Scholz; Josef Wachtveitl; Dotsch Volker Dötsch; Frank Bernhard; Clemens Glaubitz (pp. 1222-1229).

For membrane protein studies, nanodiscs have been shown to hold great potential in terms of preparing soluble samples while maintaining a lipid environment. Here, we describe the differences in lipid order and protein dynamics in MSP1 nanodiscs compared to lamellar preparations by solid-state NMR. For DMPC, an increase of the dipolar C-H lipid acyl chain order parameters in nanodiscs is observed in both gel- and liquid crystalline phases. Incorporating proteorhodopsin in these nanodiscs resulted in a significantly longer rotating frame spin-lattice relaxation time for¹³C leerzeichen and better cross polarisation efficiency due to restricted protein dynamics. A comparison of¹³C–¹³C correlation spectra revealed no structural differences. The incorporation of proteorhodopsin into nanodiscs has been optimised with respect to detergent and to protein/scaffold protein/lipid stoichiometries. Its functional state was probed by time-resolved optical spectroscopy revealing only minor differences between lamellar and nanodisc preparations. Our observations show remarkable dynamic effects between membrane proteins, lipids and scaffold protein. The potential use of nanodiscs for solid-state NMR applications is discussed.Display Omitted► Incorporation of proteorhodopsin into nanodiscs has been optimized. ► An increased order of lipid chains in nanodiscs compared to liposomes was found. ► The dynamics of proteorhodopsin is restricted in nanodiscs compared to liposomes. ► Structure and function of proteorhodopsin in nanodiscs are not altered.

Keywords: Nanodiscs; Membrane proteins; Solid-state NMR; Proteorhodopsin; Retinal proteins; Flash photolysis

Glutamate efflux mediated by Corynebacterium glutamicum MscCG, Escherichia coli MscS, and their derivatives by Michael Becker; Borngen Kirsten Börngen; Takeshi Nomura; Andrew R. Battle; Kay Marin; Boris Martinac; Kramer Reinhard Krämer (pp. 1230-1240).

Corynebacterium glutamicum is used in microbial biotechnology for the production of amino acids, in particular glutamate. The mechanism of glutamate excretion, however, is not yet fully understood. Recently, evidence was provided that the NCgl1221 gene product from C. glutamicum ATCC 13869, a MscS-type mechanosensitive efflux channel, is responsible for glutamate efflux [1]. The major difference of NCgl1221 and the homologous protein MscCG of C. glutamicum ATCC 13032 from Escherichia coli MscS and most other MscS-type proteins is the presence of an additional, 247 amino acid long C-terminal domain. By topology analysis, we show that this domain in MscCG carries a transmembrane segment. We have generated selected C-terminal truncations of MscCG, gain-of-function and loss-of-function constructs of both E. coli MscS and C. glutamicum MscCG, as well as fusion constructs of the two proteins. These mutant proteins were investigated for mechanosensitive efflux, MS channel activity, glutamate excretion and their impact on membrane potential. We provide evidence that the channel domain of MscCG mediates glutamate efflux in response to penicillin treatment, and that the E. coli MscS channel is to some extent able to function in a similar manner. We further show that the C-terminal domain of MscCG has a significant impact for function and/or regulation of MscCG. Significantly, a positive effect on glutamate efflux of the C-terminal extension of MscCG from C. glutamicum was also observed when fused to the E. coli MscS channel.Display Omitted► The channel part of MscCG from C. glutamicum mediates glutamate efflux. ► The C-terminal domain of MscCG is shown to regulate channel gating. ► Fusion of the C-terminal domain to channel domains results in glutamate efflux. ► This domain is also able to regulate heterologous channels (MscS from E. coli). ► Truncation of the C-terminal domain alters gating but not channel conductivity.

Keywords: Corynebacterium; Glutamate excretion; Mechanosensitive channel; Efflux; MscS; MscCG

Membrane properties revealed by spatiotemporal response to a local inhomogeneity by Anne-Florence Bitbol; Jean-Baptiste Fournier (pp. 1241-1249).

We study theoretically the spatiotemporal response of a lipid membrane submitted to a local chemical change of its environment, taking into account the time-dependent profile of the reagent concentration due to diffusion in the solution above the membrane. We show that the effect of the evolution of the reagent concentration profile becomes negligible after some time. It then becomes possible to extract interesting properties of the membrane response to the chemical modification. We find that a local density asymmetry between the two monolayers relaxes by spreading diffusively in the whole membrane. This behavior is driven by intermonolayer friction. Moreover, we show how the ratio of the spontaneous curvature change to the equilibrium density change induced by the chemical modification can be extracted from the dynamics of the local membrane deformation. Such information cannot be obtained by analyzing the equilibrium vesicle shapes that exist in different membrane environments in light of the area-difference elasticity model.Display Omitted► We study theoretically the response of a membrane to a local chemical perturbation. ► Chemical perturbations affect the spontaneous curvature and the equilibrium density. ► The ratio of these two effects can be inferred from the membrane dynamical response. ► Local asymmetric density perturbations relax by spreading diffusively. ► This diffusive spreading is controlled by intermonolayer friction.

Keywords: Membrane dynamics; Local perturbation; Chemical modification; Area-difference elasticity; Intermonolayer friction

The effect of oxidative stress on the membrane dipole potential of human red blood cells by S.A. Jewell; P.G. Petrov; C.P. Winlove (pp. 1250-1258).

The membrane dipole potential ( ψ_d) is an important biophysical determinant of membrane function and a sensitive indicator of lipid organisation. In this study we have used the environmentally sensitive probe di-8-anepps to explore the effects of oxidative stress on the membrane dipole potential of human erythrocytes. Cells suspended in 0.15mM phosphate buffered saline containing 0.1mg/ml albumin maintained a mean value for ψ_d of 270 (±20) mV over the course of 1hour. In the presence of 0.4mM cumene hydroperoxide there was an increase in ψ_d of 14 (±7)%, accompanied by a decrease in cell diameter of ~14 (±2)%. Exposure of the cells to 0.4mM hydrogen peroxide caused ψ_d to decrease by 13 (±8)% at the centre of the cell and 8 (±5)% at the edge whilst the diameter remained constant. In both cases the changes were equivalent to a change in transmembrane electric field of a magnitude of ~10MVm−¹, sufficient to influence membrane function. Raman microspectrometry supported the conclusion that cumene exerts its effect primarily on membrane lipids whilst hydrogen peroxide causes the formation of spectrin–haemoglobin complexes which stiffen the membrane.Display Omitted► The membrane dipole potential varies with curvature in normal red blood cells ► Effect of oxidative stress on membrane dipole potential is dependent on ROS present ► Hydrogen peroxide causes cell stiffening and decreased membrane dipole potential ► Cumene hydroperoxide causes cell shrinkage and increased membrane dipole potential

Keywords: Oxidative stress; Membrane dipole potential; Lipid packing; Erythrocytes; Di-8-ANEPPS

Molecular dynamics study of lipid bilayers modeling the plasma membranes of normal murine thymocytes and leukemic GRSL cells by Yoshimichi Andoh; Susumu Okazaki; Ryuichi Ueoka (pp. 1259-1270).

Molecular dynamics (MD) calculations for the plasma membranes of normal murine thymocytes and thymus-derived leukemic GRSL cells in water have been performed under physiological isothermal–isobaric conditions (310.15K and 1atm) to investigate changes in membrane properties induced by canceration. The model membranes used in our calculations for normal and leukemic thymocytes comprised 23 and 25 kinds of lipids, respectively, including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, lysophospholipids, and cholesterol. The mole fractions of the lipids adopted here were based on previously published experimental values. Our calculations clearly showed that the membrane area was increased in leukemic cells, and that the isothermal area compressibility of the leukemic plasma membranes was double that of normal cells. The calculated membranes of leukemic cells were thus considerably bulkier and softer in the lateral direction compared with those of normal cells. The tilt angle of the cholesterol and the conformation of the phospholipid fatty acid tails both showed a lower level of order in leukemic cell membranes compared with normal cell membranes. The lateral radial distribution function of the lipids also showed a more disordered structure in leukemic cell membranes than in normal cell membranes. These observations all show that, for the present thymocytes, the lateral structure of the membrane is considerably disordered by canceration. Furthermore, the calculated lateral self-diffusion coefficient of the lipid molecules in leukemic cell membranes was almost double that in normal cell membranes. The calculated rotational and wobbling autocorrelation functions also indicated that the molecular motion of the lipids was enhanced in leukemic cell membranes. Thus, here we have demonstrated that the membranes of thymocyte leukemic cells are more disordered and more fluid than normal cell membranes.Display Omitted► First molecular dynamics calculation for the real plasma membranes ► Membranes of the normal and leukemic thymocytes have been investigated ► Direct measurements of structure and dynamics of the membranes at a molecular level ► Changes in membrane physical properties induced by canceration has been clarified. ► The membrane of the leukemic cells is more disordered and fluid than the normal one.

Keywords: Plasma membrane; Cancer; Thymocyte; Molecular dynamics calculation; Structure; Fluidity

Investigation of interfacial behavior of glycyrrhizin with a lipid raft model via a Langmuir monolayer study by Seiichi Sakamoto; Hiromichi Nakahara; Takuhiro Uto; Yukihiro Shoyama; Osamu Shibata (pp. 1271-1283).

An interaction of glycyrrhizin (GC) with a lipid raft biomembrane model that consisted of N-palmitoyl-d- erythro-sphingosylphosphorylcholine (PSM), 1,2-dioleoyl- sn-glycero-3-phosphocholine (DOPC), and cholesterol (CHOL) was systematically studied using the Langmuir monolayer technique. To construct the lipid raft model, the surface pressure ( π)–molecular area ( A) and surface potential (Δ V)– A isotherms for three-component (PSM/DOPC/CHOL) systems on 0.02M Tris buffer with 0.13M NaCl (pH7.4) were primarily measured by changing their compositions. Thermodynamic and interaction parameters for binary PSM/DOPC and PSM/CHOL systems revealed that PSM interacts more strongly with CHOL than with DOPC. In addition, a morphological analysis performed with Brewster angle microscopy (BAM) and fluorescence microscopy (FM) revealed an optimal ratio of PSM/DOPC/CHOL (1/1/1, by mole) as a model of lipid rafts. Second, the interaction of GC with the ternary PSM/DOPC/CHOL monolayers was investigated on Tris buffer solutions containing different GC concentrations (1, 5, 10, 25, and 50μM). In BAM and FM images, microdomains were found to become smaller by increasing the GC concentration in the subphase, suggesting that GC regulates the size of raft domains, which provide dynamic scaffolding for numerous cellular processes. More interestingly, the distinctive GC striped regions were formed at the interface at 50μM, which shows that GC divides the ternary monolayer into pieces. This phenomenon was observed only in the presence of CHOL in the monolayer. These results suggest that CHOL plays an essential role in the interaction with GC, which results in one of the major activities associated with saponins' membrane disruption.Display Omitted► Lipid raft domains (PSM/DOPC/CHOL (1/1/1)) become smaller by interaction with GC. ► The membrane disruption by GC is visually observed in the monolayer study. ► LE network of lipid raft domains is promoted by increasing the amount of GC.

Keywords: Glycyrrhizin; Langmuir monolayer; Lipid raft; Membrane disruption; Saponin

Molecular origin of VDAC selectivity towards inorganic ions: A combined molecular and Brownian dynamics study by Eva-Maria Krammer; Homble Fabrice Homblé; Prevost Martine Prévost (pp. 1284-1292).

The voltage-dependent anion channel (VDAC) serves as the major pore for metabolites and electrolytes in the outer mitochondrial membrane. To refine our understanding of ion permeation through this channel we performed an extensive Brownian (BD) and molecular dynamics (MD) study on the mouse VDAC isoform 1 wild-type and mutants (K20E, D30K, K61E, E158K and K252E). The selectivity and the conductance of the wild-type and of the variant channels computed from the BD trajectories are in agreement with experimental data. The calculated selectivity is shown to be very sensitive to slight conformational changes which may have some bearing on the variability of the selectivity values measured on the VDAC open state. The MD and BD free energy profiles of the ion permeation suggest that the pore region comprising the N-terminal helix and the barrel band encircling it predominantly controls the ion transport across the channel. The overall 12μs BD and 0.9μs MD trajectories of the mouse VDAC isoform 1 wild-type and mutants feature no distinct pathways for ion diffusion and no long-lived ion–protein interactions. The dependence of ion distribution in the wild-type channel with the salt concentration can be explained by an ionic screening of the permanent charges of the protein arising from the pore. Altogether these results bolster the role of electrostatic features of the pore as the main determinant of VDAC selectivity towards inorganic anions.Display Omitted► Ions neither follow distinct pathways nor bind specifically in VDAC wt and mutants. ► The pore region about half way the channel markedly influences VDAC selectivity. ► The charge distribution of the channel controls ion selectivity of the pore. ► The calculated ion selectivity is sensitive to slight conformational changes.

Keywords: Abbreviations; BD; Brownian dynamics; hVDAC1; human VDAC isoform 1; MD; molecular dynamics; mVDAC1; mouse VDAC isoform 1; NMR3; hVDAC1 NMR structure conformation #3; N; _Cl; -; time-averaged number of chloride; N; _K; ⁺; time-averaged number of potassium; PNP; Poisson–Nernst–Planck; RMSD; root-mean square derivation; RMSF; root-mean square fluctuation; yVDAC; yeast VDAC; ΔV; _rev; difference between experimental and calculated average reversal potential valuesVDAC; Nanopore; Ion channel; Electrophysiology; Molecular dynamics; Brownian dynamics

Optimising the combination of thermostabilising mutations in the neurotensin receptor for structure determination by Yoko Shibata; Jelena Gvozdenovic-Jeremic; James Love; Brian Kloss; Jim F. White; Reinhard Grisshammer; Christopher G. Tate (pp. 1293-1301).

Conformational thermostabilisation of G protein-coupled receptors is a successful approach for their structure determination. We have recently determined the structure of a thermostabilised neurotensin receptor NTS1 in complex with its peptide agonist and here we describe the strategy for the identification and combination of the 6 thermostabilising mutations essential for crystallisation. First, thermostability assays were performed on a panel of 340 detergent-solubilised Ala/Leu NTS1 mutants and the best 16 thermostabilising mutations were identified. These mutations were combined pair-wise in nearly all combinations (119 out of a possible 120 combinations) and each mutant was expressed and its thermostability was experimentally determined. A theoretical stability score was calculated from the sum of the stabilities measured for each double mutant and applied to develop 24 triple mutants, which in turn led to the construction of 14 quadruple mutants. Use of the thermostability data for the double mutants to predict further mutant combinations resulted in a greater percentage of the triple and quadruple mutants showing improved thermostability than if only the thermostability data for the single mutations was considered. The best quadruple mutant (NTS1-Nag36k) was further improved by including an additional 2 mutations (resulting in NTS1-GW5) that were identified from a complete Ala/Leu scan of Nag36k by testing the thermostability of the mutants in situ in whole bacteria. NTS1-GW5 had excellent stability in short chain detergents and could be readily purified as a homogenous sample that ultimately allowed crystallisation and structure determination.Display Omitted► Methodology for optimal combination of thermostabilisation mutations is presented. ► Neurotensin-bound NTS1 was thermostabilised by 6 mutations (NTS1-GW5). ► NTS1-GW5 was readily purified, and has subsequently allowed structure determination.

Keywords: Neurotensin receptor; Thermostability; Membrane protein; Structure

Phase behavior and domain size in sphingomyelin-containing lipid bilayers by Robin S. Petruzielo; Frederick A. Heberle; Paul Drazba; John Katsaras; Gerald W. Feigenson (pp. 1302-1313).

Membrane raft size measurements are crucial to understanding the stability and functionality of rafts in cells. The challenge of accurately measuring raft size is evidenced by the disparate reports of domain sizes, which range from nanometers to microns for the ternary model membrane system sphingomyelin (SM)/1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC)/cholesterol (Chol). Using Förster resonance energy transfer (FRET) and differential scanning calorimetry (DSC), we established phase diagrams for porcine brain SM (bSM)/dioleoyl- sn-glycero-3-phosphocholine (DOPC)/Chol and bSM/POPC/Chol at 15 and 25°C. By combining two techniques with different spatial sensitivities, namely FRET and small-angle neutron scattering (SANS), we have significantly narrowed the uncertainty in domain size estimates for bSM/POPC/Chol mixtures. Compositional trends in FRET data revealed coexisting domains at 15 and 25°C for both mixtures, while SANS measurements detected no domain formation for bSM/POPC/Chol. Together these results indicate that liquid domains in bSM/POPC/Chol are between 2 and 7nm in radius at 25°C: that is, domains must be on the order of the 2–6nm Förster distance of the FRET probes, but smaller than the ~7nm minimum cluster size detectable with SANS. However, for palmitoyl SM (PSM)/POPC/Chol at a similar composition, SANS detected coexisting liquid domains. This increase in domain size upon replacing the natural SM component (which consists of a mixture of chain lengths) with synthetic PSM, suggests a role for SM chain length in modulating raft size in vivo.Display Omitted► FRET and DSC establish phase diagrams for bSM/DOPC/Chol and bSM/POPC/Chol at 25°C. ► FRET and SANS reveal liquid nanodomains with a 2–6nm radius for bSM/POPC/Chol. ► SM/POPC/Chol nanodomains are larger for synthetic PSM than for natural bSM.

Keywords: Abbreviations; SM; sphingomyelin; bSM; porcine brain SM; PSM; palmitoyl SM; dPSM; deuterated PSM, PSM-d31; eSM; egg SM; SSM; stearoyl SM; DOPC; dioleoyl-; sn; -glycero-3-phosphocholine; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; dPOPC; deuterated POPC, POPC-d31; Chol; cholesterol; DHE; dehydroergosterol; BoDIPY-PC; 16:0,BoDIPY-PC; LR-DOPE; lissamine rhodamine 18:1,18:1-PE; tPA; trans; -parinaric acid; Lo; liquid-ordered; Ld; liquid-disordered; high-Tm; high melting; low-Tm; low melting; Lβ; gel; Pβ′; tilted ripple phase; RSE; rapid solvent exchange; GUVs; giant unilamellar vesicles; MLVs; multilamellar vesicles; ULVs; unilamellar vesicles; FRET; Förster resonance energy transfer; R; ₀; Förster distance; SAE; sensitized acceptor emission; REE; region of enhanced efficiency; RRE; region of reduced efficiency; DSC; differential scanning calorimetry; SANS; small-angle neutron scattering; q; scattering vector; I; scattering intensity; 2θ; scattering angle; λ; neutron wavelength; SDD; sample-to-detector distance; SLD; scattering length density; Q; =; ∫; Iq; ²; dq; total scattered intensity; α; domain area fraction; Kp; partition coefficient; MD; molecular dynamicsSphingomyelin; Lipid bilayer; Coexisting liquid phases; Raft; Small-angle neutron scattering; Nanodomain

Membrane fusion and vesicular transformation induced by Alzheimer's amyloid beta by Mun'delanji C. Vestergaard; Masamune Morita; Tsutomu Hamada; Masahiro Takagi (pp. 1314-1321).

Amyloid beta (Aβ) peptides, produced through endo-proteolytic cleavage of amyloid precursor protein, are thought to be involved in the death of neural cells in Alzheimer's disease (AD). Although the mechanisms are not fully known, it has been suggested that disruption of cellular activity due to Aβ interactions with the cell membrane may be one of the underlying causes. Here in, we have investigated the interaction between Aβ-42 and biomimetic lipid membranes and the resulting perturbations in the lipid vesicles. We have shown that Aβ oligomeric species localized closer to the membrane surface. Localization of the fibrillar species of Aβ-42, although varied, was not as closely associated with the membrane surface. We have demonstrated that the presence of Aβ-42 leads to an increase in membrane surface area, inducing lipid temporal vesicular transformation. Furthermore, we have unequivocally shown that Aβ-peptides mediate membrane fusion. Although membrane fusion induced by Aβ has been hypothesized/proposed, this is the first time it has been visually captured. This fusion may be one of the mechanisms behind the membrane increase in surface area and the resulting vesicular transformation. We have shown that the longer ‘amyloidogenic’ isoform causes vesicular transformation more readily, and has a higher membrane fusogenic potential than Aβ-40. Although not core to this study, it is hugely interesting to observe the high agreement between membrane dynamics and the reported amyloidogenicity of the peptides and aggregation species opening up the potential role of vesicular dynamics for profiling and biosensing of Aβ-induced neuro-toxicity.Display Omitted► Aβ oligomeric species localized on membrane surface, but fibrils floated in an aqueous solution. ► Aβ leads to an increase in membrane surface area, inducing lipid temporal vesicular transformation. ► Aβ mediates membrane fusion.

Keywords: Amyloid beta (Aβ); Membrane dynamics; Membrane fusion; Amyloid beta species; Localization

Quantification of propidium iodide delivery using millisecond electric pulses: Experiments by Mohamed M. Sadik; Jianbo Li; Jerry W. Shan; David I. Shreiber; Hao Lin (pp. 1322-1328).

The transport mechanisms in electroporation-mediated molecular delivery are experimentally investigated and quantified. In particular, the uptake of propidium iodide (PI) into single 3T3 fibroblasts is investigated with time- and space-resolved fluorescence microscopy, and as a function of extracellular buffer conductivity. During the pulse, both the peak and the total integrated fluorescence intensity exhibit an inverse correlation with extracellular conductivity. This behavior can be explained by an electrokinetic phenomenon known as Field-Amplified Sample Stacking (FASS). Furthermore, the respective contributions from electrophoresis and diffusion have been quantified; the former is shown to be consistently higher than the latter for the experimental conditions considered. The results are compared with a compact model to predict electrophoresis-mediated transport, and good agreement is found between the two. The combination of the experimental and modeling efforts provides an effective means for the quantitative diagnosis of electroporation.Display Omitted► Quantified electrophoretic and diffusive delivery contributions for electroporation ► Determined mechanism leading to inverse relation between delivery and conductivity ► Validated a compact model that calculates molecular delivery via electrophoresis ► Discovered that cell swelling is more significant for higher buffer conductivities

Keywords: Electroporation; Electrophoresis; Field Amplified sample stacking; Molecular delivery; Extracellular conductivity

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BBA - Biomembranes (v.1828, #4)

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BBA - Biomembranes (v.1828, #4)