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BBA - Biomembranes (v.1808, #8)

Editorial Board (pp. i).

Mechanics and dynamics of triglyceride-phospholipid model membranes: Implications for cellular properties and function by Kirsi I. Pakkanen; Lars Duelund; Klaus Qvortrup; Jan S. Pedersen; John H. Ipsen (pp. 1947-1956).

We demonstrate here that triolein alters the mechanical properties of phospholipid membranes and induces extraordinary conformational dynamics. Triolein containing membranes exhibit fluctuations up to size range of 100μm and with the help of these are e.g. able to squeeze through narrow passages between neighbouring structures. Triolein–phosphatidylcholine membranes were found to have bending rigidity significantly lower than that of corresponding pure phosphatidylcholine membrane. Moreover, the triolein containing membranes were found to be reluctant to fuse, which is in good accordance with larger lamellar distances observed in the TOPOPC membranes. These findings suggest repulsion between adjacent membranes. We provide a comprehensive discussion on the possible explanations for the observed mechanics and dynamics in the TOPOPC system and on their potential cellular implications.► Triolein induces unique mechanics and dynamics in PC membranes. ► Shape dynamics and structure formation of membranes with triolein are altered. ► Bending elasticity of the membrane is significantly lowered by triolein. ► The observed membrane alterations may have significance for cell plasma membranes.

Keywords: Triglyceride; Phospholipid membrane; Membrane conformational dynamics; Membrane mechanics

Choosing membrane mimetics for NMR structural studies of transmembrane proteins by Dror E. Warschawski; Alexandre A. Arnold; Maïwenn Beaugrand; Andrée Gravel; Étienne Chartrand; Isabelle Marcotte (pp. 1957-1974).

The native environment of membrane proteins is complex and scientists have felt the need to simplify it to reduce the number of varying parameters. However, experimental problems can also arise from oversimplification which contributes to why membrane proteins are under-represented in the protein structure databank and why they were difficult to study by nuclear magnetic resonance (NMR) spectroscopy. Technological progress now allows dealing with more complex models and, in the context of NMR studies, an incredibly large number of membrane mimetics options are available. This review provides a guide to the selection of the appropriate model membrane system for membrane protein study by NMR, depending on the protein and on the type of information that is looked for. Beside bilayers (of various shapes, sizes and lamellarity), bicelles (aligned or isotropic) and detergent micelles, this review will also describe the most recent membrane mimetics such as amphipols, nanodiscs and reverse micelles. Solution and solid-state NMR will be covered as well as more exotic techniques such as DNP and MAOSS.► Several membrane mimetics available for membrane protein NMR structure determination. ► Biological and experimental requirements dictate the choice of a membrane mimetics. ► Model membranes are slow or fast tumbling and amenable to solution or solid-state NMR. ► Most common model membranes are lipid bilayers, bicelles and detergent micelles. ► Most recent membrane mimetics include amphipols, nanodiscs and reverse micelles.

Keywords: Abbreviations; AAO; anodic aluminium oxide; c7-DHPC; 1,2-heptanoyl-1-; sn; -glycero-3-phosphocholine; CMC; critical micelle concentration; CMT; critical micelle temperature; CTAB; cetyl trimethylammonium bromide; DBBPC; 1-dodecanoyl-2-(4-(4-biphenyl)butanoyl)-; sn; -glycero-3-phosphocholine; DDM; dodecylmaltoside; DHAB; dihexadecyldimethylammonium bromide; DHPC; 1,2-hexanoyl-1-; sn; -glycero-3-phosphocholine; DLPC; 1,2-dilauroyl-; sn; -glycero-3-phosphocholine; DM; decylmaltoside; DMPA; 1,2-dimyristoyl-; sn; -glycero-3-phosphate; DMPC; 1,2-myristoyl-1-; sn; -glycero-3-phosphocholine; DMPG; 1,2-dimyristoyl-; sn; -glycero-3-phospho-(1′-rac-glycerol); DMPE; 1,2-myristoyl-1-; sn; -glycero-3-phosphoethanolamine; DMPS; 1,2-dimyristoyl-; sn; -glycero-3-phospho-L-serine; DMSO; dimethyl sulfoxide; DNP; dynamic nuclear polarization; DOPC; 1,2-dioleoyl-; sn; -glycero-3-phosphocholine; DOPG; 1,2-dioleoyl-; sn; -glycero-3-phospho-(1′-; rac; -glycerol); DOPE; 1,2-oleoyl-; sn; -glycero-3-phosphoethanolamine; DOPS; 1,2-dioleoyl-; sn; -glycero-3-phospho-L-serine); DOTAP; 1,2-dioleoyl-3-trimethylammonium-propane; DPC; n; -dodecylphosphocholine; DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phosphocholine; DPPG; 1,2-dipalmitoyl-; sn; -glycero-3-phospho-(1′-; rac; -glycerol); DSPC; 1,2-distearoyl-; sn; -glycero-3-phosphocholine; GUV; giant unilamellar vesicle; HDL; high-density lipoprotein; HIMSELF; heteronuclear isotropic mixing leading to spin exchange via the local field; HSQC; heteronuclear single quantum coherence; LOPG; 1-oleoyl-2-hydroxy-; sn; -glycero-3-phospho-(1'-rac-glycerol); LPPG; 1-palmitoyl-2-hydroxy-; sn; -glycero-3-phospho-(1'-rac-glycerol); LUV; large unilamellar vesicle; MAOSS; magic angle oriented sample spinning; MAS; magic-angle spinning; MLV; multilamellar vesicle; MP; membrane protein; MSP; membrane scaffold proteins; NLP; nanolipoprotein; NMDA; N; -methyl-; d; -aspartate; NMR; nuclear magnetic resonance; NOESY; nuclear overhauser effect spectroscopy; OG; octylglucoside; PDC; protein–detergent complex; PEEK; polyetheretherketone; PET; polyethelene terephtalate; PISEMA; polarization inversion spin exchange at magic angle; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; POPG; palmitoyl-2-oleoyl-; sn; -glycero-3-phospho-(1′-rac-glycerol); PSC; protein–surfactant complex; PSPC; 1-palmitoyl-2-stearoyl-1-; sn; -glycero-3-phosphocholine; R; ²; rotational resonance; REDOR; rotational echo double resonance; SDS; sodium dodecylsulfate; SUV; small unilamllar vesicle; TBBPC; 1-tetradecanoyl-2-(4-(4-biphenyl)butanoyl)-; sn; -glycero-3-phosphocholine; TFE; 2,2,2-trifluoroethanol; T; _m; melting temperature; TROSY; transverse relaxation optimized spectroscopyMembrane protein; NMR; Membrane mimetics; Detergents; Lipids; Protein structure

Conformational and membrane interaction studies of the antimicrobial peptide alyteserin-1c and its analogue [E4K]alyteserin-1c by Anusha P. Subasinghage; Donal O'Flynn; J. Michael Conlon; Chandralal M. Hewage (pp. 1975-1984).

Alyteserin-1c (GLKEIFKAGLGSLVKGIAAHVAS.NH₂), first isolated from skin secretions of the midwife toad Alytes obstetricans, shows selective growth-inhibitory activity against Gram-negative bacteria . The structures of alyteserin-1c and its more potent and less haemolytic analogue [E4K]alyteserin-1c were investigated in various solution and membrane mimicking environments by proton NMR spectroscopy and molecular modelling. In aqueous solution, the peptide displays a lack of secondary structure but, in a 2,2,2-trifluoroethanol (TFE- d₃)-H₂O solvent mixture, the structure is characterised by an extended alpha helix between residues Leu² and Val²¹. Solution structural studies in the membrane mimicking environments, sodium dodecyl sulphate (SDS), dodecylphosphocholine (DPC), and 1,2-dihexanoyl-sn-glycero-3-phosphatidylcholine (DHPC) micelles, indicate that these peptides display an alpha helical structure between residues Lys³ and Val²¹. Positional studies of the peptides in SDS, DPC and DHPC media show that the N-terminal and central residues lie inside the micelle while C-terminal residues beyond Ala¹⁹ do not interact with the micelles.► The solution structure of alyteserin-1c is studied by proton NMR spectroscopy. ► Peptide shows a clear full-length helical structure throughout the peptide chain. ► This paper also describes the peptide interactions with the micellular media.

Keywords: Abbreviations; DHPC; 1,2-dihexanoyl-sn-glycero-3-phosphatidylcholine; DPC; dodecylphosphocholine; DSA; doxyl stearic acid; NMR; nuclear magnetic resonance; NOE; nuclear Overhauser effect; NOESY; nuclear Overhauser effect spectroscopy; SDS; sodium dodecyl sulphate; TFE; trifluroethanol; TOCSY; total correlation spectroscopy; RMSD; root mean square deviationAlyteserin; Antimicrobial; AMP; NMR; Molecular modelling; Positional studies

Ionization properties of mixed lipid membranes: A Gouy–Chapman model of the electrostatic–hydrogen bond switch by Demmelash H. Mengistu; Edgar E. Kooijman; Sylvio May / (pp. 1985-1992).

The dissociation state of phosphatidic acid (PA) in a lipid bilayer is governed by the competition of proton binding and formation of a hydrogen bond through a mechanism termed the electrostatic–hydrogen bond switch. This mechanism has been suggested to provide the basis for the specific recognition of PA by proteins. Even in bare lipid bilayers the electrostatic–hydrogen bond switch is present if the membrane contains lipids like phosphatidylethanolamine that act as hydrogen bond donors. In this case, the dissociation state (p K_a) of PA depends strongly on membrane composition. In the present work we incorporate the electrostatic–hydrogen bond switch mechanism into the Gouy–Chapman model for a membrane that is composed of PA and a hydrogen bond-donating zwitterionic lipid. To this end, our model integrates into the Gouy–Chapman approach a recently suggested electrostatic model for zwitterionic lipids. Hydrogen bond formation is incorporated phenomenologically as an additional non-electrostatic interaction between the phosphomonoester headgroup of PA and the zwitterionic lipid headgroup. We express the energetics of the composite membrane in terms of a free energy functional whose minimization leads to a modified non-linear Poisson–Boltzmann equation that we have solved numerically. Our calculations focus on the influence of the membrane environment on the dissociation state of PA. This influence can be expressed as a shift of the second p K_a of PA, which we calculate as function of membrane composition, following experimental observation. The shift is large and negative if PA is the minor component in the membrane, and it changes over four pH units as function of the mole fraction of PA in the membrane. In contrast, the shift of the second p K_a of PA remains small and is always positive if the zwitterionic lipid is unable to act as hydrogen bond donor. Hence, we find that the electrostatic–hydrogen bond switch mechanism regulates the dissociation state of PA with much greater sensitivity than would be possible based on a pure electrostatic regulation through the membrane potential.► Dissociation state of phosphatidic acid in lipid membrane is modeled. ► Gouy–Chapman model is extended to zwitterionic lipids. ► Electrostatic–hydrogen bond switch mechanism induces large shift in p K_a. ► Shift is much larger than would be possible based on electrostatics alone.

Keywords: Lipid; Membrane; Hydrogen bond; Electrostatics; Gouy-Chapman; Poisson-Boltzmann; Phosphatidic acid

Genistein and tyrphostin AG556 inhibit inwardly-rectifying Kir2.1 channels expressed in HEK 293 cells via protein tyrosine kinase inhibition by De-Yong Zhang; Wei Wu; Xiu-Ling Deng; Chu-Pak Lau; Gui-Rong Li (pp. 1993-1999).

Previous studies reported the controversial effects that protein tyrosine kinase (PTK) inhibition could induce an up-regulation or down-regulation of Kir2.1 current. The present study investigates how the recombinant human Kir2.1 channels are regulated by PTKs using whole-cell patch voltage-clamp, immunoprecipitation and Western blot, and mutagenesis approaches. We found that hKir2.1 current was reversibly inhibited by the broad spectrum PTK inhibitor genistein and the highly selective EGFR (epidermal growth factor receptor) kinase inhibitor AG556 in a concentration-dependent manner. The inhibition of hKir2.1 channels by genistein or AG556 was countered by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate. Immunoprecipitation and Western blot analysis revealed that tyrosine phosphorylation level of Kir2.1 channels was reduced by genistein or AG556, and the reduction was significantly antagonized by orthovanadate. The mutation of Y242 dramatically reduced the inhibitory response to AG556. The results obtained in this study demonstrate that hKir2.1 channels are down-regulated by PTK inhibition, suggesting that EGFR kinase participates in the modulation of human cardiac excitability.► The EGFR kinase inhibitors genistein and AG556 suppress Kir2.1 current. ► The inhibitory effect is countered by the PTP inhibitor orthovanadate. ► The Kir2.1 reduction is related to the inhibition of tyrosine phosphorylation.

Keywords: Kir2.1; Inward rectifier K; ⁺; channel; Protein tyrosine kinase; Epidermal growth factor receptor kinase

All-or-none membrane permeabilization by fengycin-type lipopeptides from Bacillus subtilis QST713 by Hiren Patel; Clemens Tscheka; Katarina Edwards; Göran Karlsson; Heiko Heerklotz (pp. 2000-2008).

The fungicidal activity of Bacillus subtilis QST713 has been utilized for the highly effective and environmentally safe protection of crops against a variety of pathogens. It is based mainly on the production of cyclic lipopeptides of the fengycin (FEs), surfactin, and iturin families. The mixed population of native FEs forms micelles which solubilize individual FEs such as agrastatin 1 (AS1) that are otherwise rather insoluble on their own. Fluorescence lifetime-based calcein efflux measurements and cryo transmission electron microscopy show that these FEs show a unique scenario of membrane permeabilization. Poor miscibility of FEs with lipid probably promotes the formation of pores in 10% of the vesicles at only≈1μM free FE and in 15% of the vesicles at 10μM. We explain why this limited, all-or-none leakage could nevertheless account for the killing of virtually all fungi whereas the same extent of graded vesicle leakage may be biologically irrelevant. Then, crystallization of AS1 and micellization of plipastatins cause a cut-off in leakage at 15% that might regulate the biological activity of FEs, protecting Bacillus and plant membranes. The fact that FE micelles solubilize only about 10mol-% fluid lipid resembles the behavior of detergent resistance.► Fungicidal Bacillus subtilis fengycin lipopeptides cause all‐or‐none membrane leakage. ► Leakage curve shows plateau due to parallel formation of micelles and crystals. ► Very active yet little soluble lipopeptide agrastatin was solubilized by others in native mix. ► Detailed leakage and partitioning data were obtained from new, fluorescence lifetime‐based assay. ► We hypothesize that all‐or‐none mechanism causes larger efflux than graded in vivo.

Keywords: Antibiotic peptide; Fungicide; Biofilm; Biosurfactant; Time resolved fluorescence spectroscopy; All-or-none leakage

The interaction of antipsychotic drugs with lipids and subsequent lipid reorganization investigated using biophysical methods by Isabel Alves; Galya Staneva; Cedric Tessier; Gilmar F. Salgado; Philippe Nuss (pp. 2009-2018).

The interaction of antipsychotic drugs (AP) with lipids and the subsequent lipid reorganization on model membranes was assessed using a combination of several complementary biophysical approaches (calorimetry, plasmon resonance, fluorescence microscopy, X-ray diffraction and molecular modeling). The effect of haloperidol (HAL), risperidone (RIS), and 9-OH-risperidone (9-OH-RIS) was examined on single lipid and mixtures comprising lipids of biological origin. All APs interact with lipids and induced membrane reorganization. APs showed higher affinity for sphingomyelin than for phosphatidylcholine. Cholesterol increased AP affinity for the lipid bilayer and led to the following AP ranking regarding affinity and structural changes: RIS >9-OH-RIS >HAL. Liquid-ordered domain formation and bilayer thickness were differentially altered by AP addition. Docking calculations helped understanding the observed differences between the APs and offer a representation of their conformation in the lipid bilayer. Present results indicate that AP drugs may change membrane compartmentalization which could differentially modulate the signaling cascade of the dopamine D2 receptor for which APs are ligands.► Antipsychotic drugs (APs) interact with lipids and induced membrane reorganization. ► APs showed higher affinity for sphingomyelin than for phosphatidylcholine. ►Cholesterol addition in lipid mixture increased APs affinity for the lipid bilayer. ► AP addition differentially modified liquid-ordered domain formation and bilayer thickness. ► Docking studies indicate that APs can differentially intercalated into the lipid bilayer.

Keywords: Antipsychotic drugs; Membrane domains; Plasmon waveguide resonance; Differential scanning calorimetry; Docking calculations; Fluorescence microscopy

Membrane binding of an acyl-lactoferricin B antimicrobial peptide from solid-state NMR experiments and molecular dynamics simulations by Tod D. Romo; Laura A. Bradney; Denise V. Greathouse; Alan Grossfield (pp. 2019-2030).

One approach to the growing health problem of antibiotic resistant bacteria is the development of antimicrobial peptides (AMPs) as alternative treatments. The mechanism by which these AMPs selectively attack the bacterial membrane is not well understood, but is believed to depend on differences in membrane lipid composition. N-acylation of the small amidated hexapeptide, RRWQWR-NH₂ (LfB6), derived from the 25 amino acid bovine lactoferricin (LfB25) can be an effective means to improve its antimicrobial properties. Here, we investigate the interactions of C6-LfB6, N-acylated with a 6 carbon fatty acid, with model lipid bilayers with two distinct compositions: 3:1 POPE:POPG (negatively charged) and POPC (zwitterionic). Results from solid-state²H and³¹P NMR experiments are compared with those from an ensemble of all-atom molecular dynamic simulations running in aggregate more than 8.6ms.²H NMR spectra reveal no change in the lipid acyl chain order when C6-LfB6 is bound to the negatively charged membrane and only a slight decrease in order when it is bound to the zwitterionic membrane.³¹P NMR spectra show no significant perturbation of the phosphate head groups of either lipid system in the presence of C6-LfB6. Molecular dynamic simulations show that for the negatively charged membrane, the peptide's arginines drive the initial association with the membrane, followed by attachment of the tryptophans at the membrane–water interface, and finally by the insertion of the C6 tails deep into the bilayer. In contrast, the C6 tail leads the association with the zwitterionic membrane, with the tryptophans and arginines associating with the membrane–water interface in roughly the same amount of time. We find similar patterns in the order parameters from our simulations. Moreover, we find in the simulations that the C6 tail can insert 1–2Å more deeply into the zwitterionic membrane and can exist in a wider range of angles than in the negatively charged membrane. We propose this is due to the larger area per lipid in the zwitterionic membrane, which provides more space for the C6 to insert and assume different orientations.► Association of small acylated peptide with model bacterial and mammalian membranes. ► Bacterial membrane shows no change in order, and binding is led by arginines. ► Mammalian membrane has decrease in order, and binding is led by C6 tail.

Keywords: acyl-peptides; Antimicrobial; Lactoferricin; Molecular dynamics; Membrane interactions; Solid-state NMR

The effect of liposomes’ surface electric potential on the uptake of hematoporphyrin by Danor Aharon; Hana Weitman; Benjamin Ehrenberg (pp. 2031-2035).

Hematoporphyrin is being used as a photosensitizer in photodynamic therapy of tumors, as well as of other clinical cases. Many classes of tetrapyrroles, including hematoporphyrin, are partitioning quite easily into the external cytoplasmic membrane as the mechanism of cellular uptake. Several chemical and physical parameters of the membrane were studied for their effect on the extent of porphyrins' partitioning. In this manuscript we report, for the first time, a quantitative analysis of the effect of the membrane's surface electric potential on the partitioning. We prepared liposomes, as membrane models, composed on zwitterionic DMPC lipid, as well as DMPC liposomes that contain a small, varying fraction of negatively charged DMPS and positively charged DOTAP. We found that indeed the surface potential had a very strong effect on the binding constant of HP, which is negatively charged at the physiological pH that was used. The trend in the apparent binding constant can be formulated and fitted with the Gouy-Chapman model of surface potential. We found that the average concentration of HP within the aqueous shell that has a thickness of the Debye layer around the liposome is determining the extent of binding in the law of mass action.► We probed the effect of membrane electric surface potential on the binding of hematoporphyrin to liposomes. ► The binding is very strongly affected by the surface potential, which was generated by the addition of charged lipid molecules. ► The trend in the binding constant can be nicely accommodated with the Gouy-Chapman model of surface potential. ► The local average concentration of hematoporphyrin in the Debye layer around the liposome is the factor which determines the equilibrium of binding.

Keywords: Debye layer; Gouy-Chapman; Hematoporphyrin; Liposome; Surface potential

Volume regulation of murine T lymphocytes relies on voltage-dependent and two-pore domain potassium channels by Nicole Bobak; Stefan Bittner; Joseph Andronic; Susanne Hartmann; Muhlpfordt Friederike Mühlpfordt; Tilman Schneider-Hohendorf; Karen Wolf; Carsten Schmelter; Gobel Kerstin Göbel; Patrick Meuth; Heiko Zimmermann; Doring Frank Döring; Erhard Wischmeyer; Thomas Budde; Heinz Wiendl; Sven G. Meuth; Vladimir L. Sukhorukov (pp. 2036-2044).

A variety of ion channels are supposed to orchestrate the homoeostatic volume regulation in T lymphocytes. However, the relative contribution of different potassium channels to the osmotic volume regulation and in particular to the regulatory volume decrease (RVD) in T cells is far from clear. This study explores a putative role of the newly identified K_2P channels (TASK1, TASK2, TASK3 and TRESK) along with the voltage-gated potassium channel K_V1.3 and the calcium-activated potassium channel K_Ca3.1 in the RVD of murine T lymphocytes, using genetic and pharmacological approaches. K_2P channel knockouts exerted profound effects on the osmotic properties of murine T lymphocytes, as revealed by reduced water and RVD-related solute permeabilities. Moreover, both genetic and pharmacological data proved a key role of K_V1.3 and TASK2 channels in the RVD of murine T cells exposed to hypotonic saline. Our experiments demonstrate a leading role of potassium channels in the osmoregulation of T lymphocytes under different conditions. In summary, the present study sheds new light on the complex and partially redundant network of potassium channels involved in the basic physiological process of the cellular volume homeostasis and extends the repertoire of potassium channels by the family of K_2P channels.► Investigation of the functional role of members of the K_2P family in volume regulation in lymphocytes ► K_V1.3 and TASK2 are mainly responsible for RVD in murine T lymphocytes ► The impact of K_2P channels on RVD has been shown to depend on the cell activation status

Keywords: Abbreviations; CGM; complete growth medium; K; _2P; two-pore-domain potassium channels; MACS; magnetic cell separation; PDMS; polydimethylsiloxane; PBS; phosphate buffered saline; RVD; regulatory volume decrease; RVI; regulatory volume increase; ShK; Stichodactyla; toxin; TRAM34; 1-[(2-Chlorophenyl)diphenylmethyl]-1H-pyrazole; Wt; wild typeCell volumetry; Regulatory volume decrease; Osmotic stress; T lymphocyte; Potassium channel; K; _2P; channels

The A391E mutation enhances FGFR3 activation in the absence of ligand by Fenghao Chen; Catherine Degnin; Melanie Laederich; William A. Horton; Kalina Hristova (pp. 2045-2050).

The A391E mutation in the transmembrane domain of fibroblast growth factor receptor 3 leads to aberrant development of the cranium. It has been hypothesized that the mutant glutamic acid stabilizes the dimeric receptor due to hydrogen bonding and enhances its ligand-independent activation. We previously tested this hypothesis in lipid bilayers and showed that the mutation stabilizes the isolated transmembrane domain dimer by −1.3°kcal/mol. Here we further test the hypothesis, by investigating the effect of the A391E mutation on the activation of full-length fibroblast growth factor receptor 3 in Human Embryonic Kidney 293T cells in the absence of ligand. We find that the mutation enhances the ligand-independent activation propensity of the receptor by −1.7°kcal/mol. This value is consistent with the observed strength of hydrogen bonds in membranes, and supports the above hypothesis.► The plasma membrane expression of the A391E mutant is lower than the wild-type FGFR3 surface expression in HEK 293T cells. ► The A391E mutation increases the activation propensity of full-length FGFR3 in the absence of ligand by –1.7 kcal/mole. ► Pathogenesis due to the A391E FGFR3 mutation is likely linked to disregulation of ligand-independent activation.

Keywords: Membrane proteins; Cell signaling

5- and 4′-Hydroxylated flavonoids affect voltage gating of single alpha-hemolysin pore by Olga S. Ostroumova; Svetlana S. Efimova; Ludmila V. Schagina (pp. 2051-2058).

Molecular mechanisms of the influence of flavonoids on the voltage gating of a single alpha-hemolysin channel in planar lipid membranes are studied. It is shown that the addition of flavonoids hydroxylated in position 5 of the A-ring and in position 4′ of the B-ring into bilayer bathing solution shifts the voltage dependence of channel switching from high- to low-conductance states to voltages nearer zero. It is concluded that the effect is likely to be attributed to a specific interaction of at least three flavonoid molecules with the voltage sensor of an alpha-hemolysin pore. Possible flavonoid binding sites and identification of amino acid residues included into the voltage sensor domain of the alpha-hemolysin channel are discussed.► Flavonoids hydroxylated in position 5 of the A-ring and in position 4' of the B-ring shifts the voltage dependence of alpha-hemolysin channel switching from high- to low-conductance states to voltages nearer zero. ► Introduction of flavonoids does not alter the conductance, lifetime and the selectivity of low conductance state of alpha-hemolysin pore. ► The effect is attributed to a specific interaction of at least three flavonoid molecules with the voltage sensor of single alpha-hemolysin pore. ► Comparison of the distances between the polar amino acid residues in the same positions of neighboring protomers and between 5-OH and 4'-OH-groups of the flavonoid molecule allows speculating that the most appropriate candidate for a possible flavonoid binding site is Thr129.

Keywords: Abbreviations; α-HL; alpha-hemolysin; HCS; high-conductance state; LCS; low-conductance state; PC; 1,2-dioleoyl-; sn; -glycero-3-phosphocholinePlanar lipid bilayers; Alpha-hemolysin channel; Channel voltage gating; Dipole potential modifiers; Flavonoids

Expression of a chloroplast ATP/ADP transporter in E. coli membranes: Behind the Mistic strategy by Aurélien Deniaud; Florent Bernaudat; Annie Frelet-Barrand; Céline Juillan-Binard; Thierry Vernet; Norbert Rolland; Eva Pebay-Peyroula (pp. 2059-2066).

Eukaryotic membrane protein expression is still a major bottleneck for structural studies. Production in E. coli often leads to low expression level and/or aggregated proteins. In the last decade, strategies relying on new fusion protein expression revealed promising results. Fusion with the amphipatic Mistic protein has been described to favor expression in E. coli membranes. Although, this approach has already been reported for a few membrane proteins, little is known about the activity of the fused proteins. We used this strategy and obtained high expression levels of a chloroplast ATP/ADP transporter from A. thaliana (NTT1) and characterized its transport properties. NTT1 fused to Mistic has a very low transport activity which can be recovered after in vivo Mistic fusion cleavage. Moreover, detailed molecular characterization of purified NTT1 mature form, NTT1 fused to Mistic or NTT1 cleaved-off from this fusion highlights the correct fold of the latter one. Therefore, considering the higher quantity of purified NTT1 mature form obtained via the Mistic fusion approach, this is a valuable strategy for obtaining quantities of pure and active proteins that are adequate for structural studies.► An ATP/ADP chloroplast transporter can be expressed in mg amounts in fusion with Mistic. ► The fusion with Mistic leads to a low activity ATP/ADP transporter. ► In vivo fusion cleavage delivers a fully active transporter. ► Biophysical characterization shows that Mistic fusion forms large oligomers. ► Well-folded NTT1 mature form can be purified after in vitro fusion cleavage.

Keywords: Membrane protein heterologous expression; Mistic fusion strategy; ATP/ADP chloroplast transporter; In vivo; fusion cleavage

Effects of a bacterial trehalose lipid on phosphatidylglycerol membranes by Antonio Ortiz; José A. Teruel; Ángeles Manresa; María J. Espuny; Marques Ana Marqués; Francisco J. Aranda (pp. 2067-2072).

Bacterial trehalose lipids are biosurfactants with potential application in the biomedical/healthcare industry due to their interesting biological properties. Given the amphiphilic nature of trehalose lipids, the understanding of the molecular mechanism of their biological action requires that the interaction between biosurfactant and membranes is known. In this study we examine the interactions between a trehalose lipid from Rhodococcus sp. and dimyristoylphosphatidylglycerol membranes by means of differential scanning calorimetry, X-ray diffraction, infrared spectroscopy and fluorescence polarization. We report that there are extensive interactions between trehalose lipid and dimyristoylphosphatidylglycerol involving the perturbation of the thermotropic gel to liquid-crystalline phase transition of the phospholipid, the increase of fluidity of the phosphatidylglycerol acyl chains and dehydration of the interfacial region of the bilayer, and the modulation of the order of the phospholipid bilayer. The observations are interpreted in terms of structural perturbations affecting the function of the membrane that might underline the biological actions of the trehalose lipid.► Trehalose lipid perturbs the phase transition of dimyristoylphosphatidylglycerol. ► The biosurfactant increases the fluidity of the phosphatidylglycerol acyl chains. ► Trehalose lipid dehydrates the interfacial region of the bilayer. ► Trehalose lipid modulates the order of the phospholipid palisade.

Keywords: Trehalose lipid; Phosphatidylglycerol; DSC; X-ray diffraction; FT-IR; Fluorescence polarization

Role of the putative N-glycosylation and PKC-phosphorylation sites of the human sodium-dependent multivitamin transporter (hSMVT) in function and regulation by Abhisek Ghosal; Veedamali S. Subramanian; Hamid M. Said (pp. 2073-2080).

The sodium-dependent multivitamin transporter (SMVT) is a major biotin transporter in a variety of tissues including the small intestine. The human SMVT (hSMVT) polypeptide is predicted to have four N-glycosylation sites and two putative PKC phosphorylation sites but their role in the function and regulation of the protein is not known and was examined in this investigation. Our results showed that the hSMVT protein is glycosylated and that this glycosylation is important for its function. Studies utilizing site-directed mutagenesis revealed that the N-glycosylation sites at positions Asn¹³⁸ and Asn⁴⁸⁹ are important for the function of hSMVT and that mutating these sites significantly reduces the V_max of the biotin uptake process. Mutating the putative PKC phosphorylation site Thr²⁸⁶ of hSMVT led to a significant decrease in the PMA-induced inhibition in biotin uptake. The latter effect was not mediated via changes in the level of expression of the hSMVT protein and mRNA or in its level of expression at the cell membrane. These findings demonstrate that the hSMVT protein is glycosylated, and that glycosylation is important for its function. Furthermore, the study shows a role for the putative PKC-phosphorylation site Thr²⁸⁶ of hSMVT in the PKC-mediated regulation of biotin uptake.► The human sodium-dependent multivitamin transporter (hSMVT) is glycosylated. ► The glycosylation Asn¹³⁸ and Asn⁴⁸⁹ are essential for hSMVT function. ► The putative PKC-phosphorylation site Thr²⁸⁶ in the hSMVT polypeptide is important in the PKC-mediated regulation of biotin uptake.

Keywords: Biotin; hSMVT; Glycosylation; PKC; Transport; Regulation

Spatial structure and dimer–monomer equilibrium of the ErbB3 transmembrane domain in DPC micelles by K.S. Mineev; N.F. Khabibullina; E.N. Lyukmanova; D.A. Dolgikh; M.P. Kirpichnikov; A.S. Arseniev (pp. 2081-2088).

In present work the interaction of two TM α-helices of the ErbB3 receptor tyrosine kinase from the ErbB or HER family (residues 639–670) was studied by means of NMR spectroscopy in a membrane-mimicking environment provided by the DPC micelles. The ErbB3 TM segment appeared to form a parallel symmetric dimer in a left-handed orientation. The interaction between TM spans is accomplished via the non-standard motif and is supported by apolar contacts of bulky side chains and by stacking of aromatic rings together with π–cation interactions of Phe and Arg side chains.The investigation of the dimer–monomer equilibrium revealed thermodynamic properties of the assembly and the presence of two distinct regimes of the dimerization at low and at high peptide/detergent ratio. It was found that the detergent in case of ErbB3 behaves not as an ideal solvent, thus affecting the dimer–monomer equilibrium. Such behavior may account for the problems occurring with the refolding and stability of multispan helical membrane proteins in detergent solutions. The example of ErbB3 allows us to conclude that the thermodynamic parameters of dimerization, measured in micelles for two different helical pairs, cannot be compared without the investigation of their dependence on detergent concentration.► We study structure and dimerization equilibrium of ErbB3 transmembrane domain in micelles. ► ErbB3 transmembrane domain forms a symmetric parallel left-handed homodimer. ► It employs nonstandard motif for helix–helix interaction. ► The apparent free energy of dimerization was measured depending on detergent concentration. ► Dodecylphosphocholine behaves as nonideal solvent for the interaction of ErbB3 helices.

Keywords: Abbreviations; TM; transmembrane; DPC; dodecylphosphocholine; tm; isolated transmembrane domain; RTK; receptor tyrosine kinase; LDAO; Lauryldimethylamine-oxide; FRET; Foerster resonance energy transfer; TFE; trifluoroethanol; TFA; trifluoroacetic acid; NOE; nuclear Overhauser effect; NOESY; NOE spectroscopy; CMC; critical micelle concentrationReceptor tyrosine kinases; Transmembrane domain; Dimerization; Structure; NMR; Dimerization constant

Erratum to “Reviewer Acknowledgment” [Biochem. Biophys. Acta 1808 (2011) iii–v] (pp. 2089-2094).

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BBA - Biomembranes (v.1808, #8)