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BBA - Biomembranes (v.1818, #12)

Editorial Board (pp. i).

Efficacy verification and microscopic observations of an anticancer peptide, CB1a, on single lung cancer cell by Feng-Sheng Kao; Yun-Ru Pan; Ray-Quen Hsu; Hueih-Min Chen (pp. 2927-2935).

In this work, we introduce a new customized anti-lung cancer peptide, CB1a, with IC₅₀ of about 25.0±1.6μM on NCI-H460 lung cancer cells. Using a multi-cellular tumor spheroid (MCTS) model, results show that CB1a is potent in preventing the growth of lung cancer tumor-like growths in vitro. Additionally, atomic force microscopy (AFM) was used to examine cell surface damage of a single cancer. The mechanism for cell death under CB1a toxicity was verified as being largely due to cell surface damage. Moreover, with a treatment dosage of CB1a at 25μM, Young's module ( E) shows that the elasticity and stiffness of cancer cell decreased with time such that the interaction time for a 50% reduction of E (IT₅₀) was about 7.0min. This new single-cell toxicity investigation using IT₅₀ under AFM assay can be used to separately verify drug efficacy in support of the traditional IC₅₀ measurement in bulk solution. These results could be of special interest to researchers engaged in new drug development.In this work, two points are presented: (1) CB1a can be developed as potential anticancer drug and (2) a new approach indicates the efficacy of CB1a. Using a multi-cellular tumor spheroid (MCTS) model, results show that CB1a is potent in preventing the growth of lung cancer tumor-like growths in vitro (Fig. A). Additionally, atomic force microscopy (AFM) was used to examine cell surface damage of a single cancer. The mechanism for cell death under CB1a toxicity was verified as being largely due to cell surface damage. Moreover, with a treatment dosage of CB1a at 25μM, Young's module ( E) shows that the elasticity and stiffness of cancer cell decreased with time such that the Interaction Time for a 50% reduction of E (IT₅₀) was about 7.0min (Fig. B). This new single-cell toxicity investigation using IT₅₀ under AFM assay can be used to separately verify drug efficacy in support of the traditional IC₅₀ measurement in bulk solution.Display Omitted► We originally observed the surface damage of a single lung cancer cell by a custom peptide using AFM. ► We created a new term” IT50” for cell-killed evaluation. ► Cross verification of drug efficacy by measuring both IT50 and IC50 was proposed for the first time.

Keywords: Abbreviations; AFM; atomic force microscopy; AMP; antimicrobial peptide; CB; cecropin B; Dox; doxorubicin; EM; electron microscopy; IT; ₅₀; interaction time (IT) for reducing 50% of; E; MCTS; Multi-cellular tumor spheroids; SI; selectivity index; SOP; standard operation procedureAtomic force microscopy; CB1a; single cell; Cancer cell; Anticancer peptide

Membrane cholesterol stabilizes the human serotonin_1A receptor by Roopali Saxena; Amitabha Chattopadhyay (pp. 2936-2942).

A number of recently solved crystal structures of G-protein coupled receptors reveal the presence of closely associated cholesterol molecules in the receptor structure. We have previously shown the requirement of membrane cholesterol in the organization, dynamics and function of the serotonin_1A receptor, a representative G‐protein coupled receptor. In this work, we explored the role of membrane cholesterol in the stability of the human serotonin_1A receptor. Analysis of sensitivity of the receptor to thermal deactivation, pH, and proteolytic digestion in control, cholesterol-depleted and cholesterol-enriched membranes comprehensively demonstrate that membrane cholesterol stabilizes the serotonin_1A receptor. We conclude that these results could have potential implications in future efforts toward crystallizing the receptor.Display Omitted► Membrane cholesterol stabilizes ligand binding of the serotonin_1A receptor. ► This is analogous to the stabilizing role of trehalose for proteins. ► These results have implications in future crystallization efforts of the receptor.

Keywords: Abbreviations; 5-HT; _1A; receptor; 5-hydroxytryptamine-1A receptor; 8-OH-DPAT; 8-hydroxy-2(di-; N; -propylamino)tetralin; BCA; bicinchoninic acid; BSA; bovine serum albumin; CAPS; 3-[cyclohexylamino]-1-propanesulfonic acid; CCM; cholesterol consensus motif; CRAC; cholesterol recognition/interaction amino acid consensus; DMPC; dimyristoyl-; sn; -glycero-3-phosphocholine; DPH; 1,6-diphenyl-1,3,5-hexatriene; GPCR; G-protein coupled receptor; MβCD; methyl-β-cyclodextrin; PMSF; phenylmethylsulfonyl fluoride; Tris; tris; -(hydroxymethyl)aminomethaneHuman serotonin; _1A; receptor; Cholesterol; Ligand binding; Thermal deactivation; Fluorescence anisotropy

Atomic force microscopy imaging of lipid rafts of human breast cancer cells by F. Orsini; A. Cremona; P. Arosio; P.A. Corsetto; G. Montorfano; A. Lascialfari; A.M. Rizzo (pp. 2943-2949).

Several studies suggest that the plasma membrane is composed of micro-domains of saturated lipids that segregate together to form lipid rafts. Lipid rafts have been operationally defined as cholesterol- and sphingolipid-enriched membrane micro-domains resistant to solubilization by non-ionic detergents at low temperatures. Here we report a biophysical approach aimed at investigating lipid rafts of MDA-MB-231 human breast cancer cells by coupling an atomic force microscopy (AFM) study to biochemical assays namely Western blotting and high performance thin layer chromatography. Lipid rafts were purified by ultracentrifugation on discontinuous sucrose gradient using extraction with Triton X-100. Biochemical analyses proved that the fractions isolated at the 5% and 30% sucrose interface (fractions 5 and 6) have a higher content of cholesterol, sphingomyelin and flotillin-1 with respect to the other purified fractions. Tapping mode AFM imaging of fraction 5 showed membrane patches whose height corresponds to the one awaited for a single lipid bilayer as well as the presence of micro-domains with lateral dimensions in the order of a few hundreds of nanometers. In addition, an AFM study using specific antibodies suggests the presence, in these micro-domains, of a characteristic marker of lipid rafts, the protein flotillin-1.Display Omitted► Lipid rafts of MDA-MB-231 cancer cells were studied by AFM and biochemical assays. ► AFM showed membrane micro-domains with lateral sizes of a few hundreds of nanometers. ► WB and HP-TLC detected a high CHOL, SM and Flot-1 content in the micro-domains. ► AFM study using specific antibodies identified Flot-1 in the micro-domains.

Keywords: Lipid rafts; Atomic force microscopy; MDA-MB-231 cancer cells

HIV-1 variants with a single-point mutation in the gp41 pocket region exhibiting different susceptibility to HIV fusion inhibitors with pocket- or membrane-binding domain by Lu Lu; Pei Tong; Xiaowen Yu; Chungen Pan; Peng Zou; Ying-Hua Chen; Shibo Jiang (pp. 2950-2957).

Enfuvirtide (T20), the first FDA-approved peptide HIV fusion/entry inhibitor derived from the HIV-1 gp41 C-terminal heptad-repeat (CHR) domain, is believed to share a target with C34, another well-characterized CHR-peptide, by interacting with the gp41 N-terminal heptad-repeat (NHR) to form six-helix bundle core. However, our previous studies showed that T20 mainly interacts with the N-terminal region of the NHR (N-NHR) and lipid membranes, while C34 mainly binds to the NHR C-terminal pocket region. But so far, no one has shown that C34 can induce drug-resistance mutation in the gp41 pocket region. In this study, we constructed pseudoviruses in which the Ala at the position of 67 in the gp41 pocket region was substituted with Asp, Gly or Ser, respectively, and found that these mutations rendered the viruses highly resistant to C34, but sensitive to T20. The NHR-peptide N36 with mutations of A67 exhibited reduced anti-HIV-1 activity and decreased α-helicity. The stability of six-helix bundle formed by C34 and N36 with A67 mutations was significantly lower than that formed by C34 and N36 with wild-type sequence. The combination of C34 and T20 resulted in potent synergistic anti-HIV-1 effect against the viruses with mutations in either N- or C-terminal region in NHR. These results suggest that C34 with a pocket-binding domain and T20 containing the N-NHR- and membrane-binding domains inhibit HIV-1 fusion by interacting with different target sites and the combinatorial use of C34 and T20 is expected to be effective against HIV-1 variants resistant to HIV fusion inhibitors.Display Omitted► T20 contains membrane-binding domain (MBD) but lacks pocket-binding domain (PBD). ► We constructed pseudoviruses with mutations of A67 in gp41 NHR pocket region. ► These mutants are sensitive to T20 lacking PBD but resistant to C34 that contains PBD. ► Combining T20 & C34 lead to synergistic effect against T20- & C34-resistant mutants. ► These suggest that T20 and C34 inhibit HIV-1 fusion by binding to different targets.

Keywords: Abbreviations; NHR; N-terminal heptad repeat; CHR; C-terminal heptad repeat; 6-HB; six-helix bundle; Env; envelope protein; HBD; heptad-repeat binding domain; PBD; pocket-binding domain; PFD; pocket-forming domain; MBD; membrane-binding domain; CD; Circular Dichroism; N-PAGE; native polyacrylamide gel electrophoresis; TCID; ₅₀; 50% tissue culture infective dose; IC; ₅₀; concentration causing 50% inhibitionHIV-1; gp41; Fusion inhibitors; Mutation; Drug-resistance; Membrane-binding

The membrane spanning domains of protein NS4B from hepatitis C virus by F. Palomares-Jerez; Henrique Nemesio; Villalain José Villalaín (pp. 2958-2966).

Determination of the membrane spanning domains of highly hydrophobic proteins from its primary structure, i.e., sequence, is cumbersome. However, transmembrane topology is better correlated with protein secondary structure than with the primary one. In this work we have determined the number and location of the transmembrane domains of the highly hydrophobic hepatitis C virus NS4B protein by studying the water-to-bilayer and water-to-interface transfer free energies of thirty-one different hepatitis C virus strains assuming that NS4B forms an α-helical wheel. Additionally, we have studied the effect of a peptide library encompassing the full length of the NS4B protein hepatitis C virus strain 1a_H77 on the phase transitions of DEPE through the use of differential scanning calorimetry. Our findings show that NS4B protein has five transmembrane domains and, as previously suggested, three interfacial segments. One of these segments, segment AH2, could behave similarly to viral pre-transmembrane segments, which would partition into and interact with the membrane and be responsible for the fluctuation of the protein between different topologies and therefore possible locations.Display Omitted► The membrane topology of the HCV NS4B protein is described. ► HCV NS4B has five transmembrane domains and possibly a sixth one. ► The sixth possible transmembrane domain of NS4B could behave as a viral pre-transmembrane segment. ► These segments should be involved in the formation of the replication complex.

Keywords: NS4B; HCV; Pre-transmembrane domain

Membrane interactions of ionic liquids: Possible determinants for biological activity and toxicity by Noga Gal; Danilo Malferarri; Sofiya Kolusheva; Paola Galletti; Emilio Tagliavini; Raz Jelinek (pp. 2967-2974).

Ionic liquids (ILs) are a class of diverse organic salts with relatively low melting points (below 100°C) which have attracted considerable interest as a promising “green” substitute for organic solvents. The broad solvation properties of ILs and their high solubility in water, however, present health risks, in particular since it was shown that many ILs exhibit cytotoxic properties. In this context, interactions of ILs with the cellular membrane are believed to constitute a primary culprit for toxicity. We present a comprehensive biophysical and microscopy study of membrane interactions of a series of ILs having different side-chain compositions and lengths, and cationic head-group structures and orientations. The experimental data reveal that the ILs studied exhibit distinct mechanisms of membrane binding, insertion, and disruption which could be correlated with their biological activities. The results indicate, in particular, that both the side chain composition and particularly the head-groups of ILs constitute determinants for membrane activity and consequent cell toxicity. This work suggests that tuning membrane interactions of ILs should be an important factor for designing future compounds with benign environmental impact.Display Omitted► Ionic liquids exhibit distinct models of membrane interactions. ► Membrane permeation and disruption can be correlated to ionic liquid toxicity. ► Both ionic liquids' side-chains, but particularly the head-group appear to contribute to membrane disruption.

Keywords: Abbreviations; PDA; polydiacetylene; DMPC; dimyristoylphosphatydilcholine; DMPG; dimyristoylphosphatidylglycerol; DSC; differential scanning calorimetry; FRET; F; ö; rster resonance energy transfer; GUV; giant vesicle; IL; ionic liquid; NBD-PE; N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)1,2-dihexadecanoyl-snglycero-3-phosphoethanolamineIonic liquid; Membrane; Membranes permeation; Giant vesicle; Polydiacetylene; FRET

Binding and reorientation of melittin in a POPC bilayer: Computer simulations by Sheeba J. Irudayam; Max L. Berkowitz (pp. 2975-2981).

We performed, using an all-atom force field, molecular dynamics computer simulations to study the binding of melittin to the POPC bilayer and its subsequent reorientation in this bilayer. The binding process involves a simultaneous folding and adsorption of the peptide to the bilayer, followed by the creation of a “U shaped” conformation. The reorientation of melittin from the parallel to the perpendicular conformation requires charged residues to cross the hydrophobic core of the bilayer. This is accomplished by a creation of defects in the bilayer that are filled out with water. The defects are caused by peptide charged residues dragging the lipid headgroup atoms along with them, as they reorient. With increased concentration of melittin water defects form stable pores; this makes it easier for the peptide N-terminus to reorient. Our results complement experimental and computational observations of the melittin/lipid bilayer interaction.Display Omitted► Melittin can adopt a shallow “U-shaped” conformation upon binding to a POPC bilayer. ► Reorientation of melittin at a P/L ratio=1/128 causes water defects in the bilayer. ► At P/L=4/128, upon reorientation, the water defects form stable toroidal pores. ► Pseudo-transmembrane conformation is an intermediate in the reorientation process.

Keywords: Antimicrobial peptides; Lipid bilayers; Pores in bilayers; Molecular dynamics simulations; Melittin

Membrane structure and interactions of peptide hormones with model lipid bilayers by Emilia Sikorska; Ilowska Emilia Iłowska; Dariusz Wyrzykowski; Anna Kwiatkowska (pp. 2982-2993).

In this work, the behavior of the neurohypophyseal hormones and their selected analogs was studied in the presence of membrane models in an attempt to correlate their activities with a distinct behavior at a level of peptide–lipid interactions. The influence of the peptides studied on the lipid acyl chain order was determined using FTIR spectroscopy. Conformational changes in the peptides upon binding to liposomes were examined using CD spectra. Attempts were also made to determine the binding parameters of the peptides to lipids using isothermal titration calorimetry (ITC). The results show unambiguously that the neurohyphophyseal hormone-like peptides interact with lipids, being a model of a eukaryotic cell membrane. Moreover, hydrophobic interactions between the peptides and liposomes are likely to determine the overall conformation of the peptide, especially below the temperature of the main phase transition (T_m). Thus, the bulky and hydrophobic nature of the residues incorporated into the N-terminal part of neurohyphophyseal hormones is an important factor for both restriction of peptide mobility and the interaction of the analog with biomembrane. In turn, above T_m, the electrostatic interactions become also relevant for the conformation of the acyclic tail of the AVP-like peptides.Display Omitted► The behavior of the AVP and OT-like peptides at a level of peptide–lipid interactions. ► The hydrophobic modification improves the peptide insertion into the lipid bilayer. ► The hydrophobic interactions between the peptides and liposomes determine the overall conformation of the peptide. ► The electrostatic interactions become relevant for the AVP-like peptides above T_m.

Keywords: Abbreviations; Adg; (S)-2-(1-adamantyl)glycine; AVP; arginine vasopressin; cis; -Apc; cis; -1-amino-4-phenylcyclohexane-1-carboxylic acid; D-Igl; D-α-2-indanylglycine; DPC; dodecylphosphocholine; DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phosphocholine; DPPG; 1,2-dipalmitoyl-; sn; -glycero-3-phosphoglycerol; Ica; indoline-2-carboxylic acid; GPCR; G protein-coupled receptor; LUV; large unilamellar vesicle; Mpa; 3-mercaptopropionic acid; MLV; multilayer vesicle; OT; oxytocin; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphoglycerol; SDS; sodium dodecyl sulfateNeurohypophyseal hormones; Model membrane; CD; FTIR; ITC

Effect of juxtamembrane tryptophans on the immersion depth of Synaptobrevin, an integral vesicle membrane protein by M. Sameer Al-Abdul-Wahid; Colin M. DeMill; Marzena B. Serwin; R. Scott Prosser; Bryan A. Stewart (pp. 2994-2999).

Proper positioning of membrane proteins in the host membrane is often critical to successful protein function. While hydrophobic considerations play a dominant role in determining the topology of a protein in the membrane, amphiphilic residues, such as tryptophan, may ‘anchor’ the protein near the water–membrane interface. The SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family of membrane proteins mediates intracellular membrane fusion. Correct positioning of the SNAREs is necessary if fusion is to occur. Synaptobrevins are integral vesicle membrane proteins that are well conserved across species. Interestingly, mammalian Synaptobrevins typically contain two adjacent tryptophans near the water-membrane interface whereas the Drosophila, neuronal-Synaptobrevin (n-Syb), contains a single tryptophan in this same region. To explore the role of these tryptophan residues in membrane positioning, we prepared a peptide containing residues 75–121 of D. melanogaster n-Syb in DPC micelles, biosynthetically labeled with 4-fluorophenylalanine and 5-fluorotryptophan for the examination by¹⁹F NMR spectroscopy. Mutations of this construct containing zero and two tryptophan residues near the water-membrane interface resulted in changes in the positioning of n-Syb in the micelle. Moreover, the addition of a second tryptophan appears to slow dynamic motions of n-Syb near the micelle–water interface. These data therefore indicate that juxtamembrane tryptophan residues are important determinants of the position of Synaptobrevin in the membrane.Display Omitted► We examine the positioning of neuronal-Synaptobrevin within micelles. ► Mutants with zero and two Trp residues are compared to wild-type (one Trp). ► Mutations of juxtamembrane tryptophan residues affect membrane positioning. ► Decreasing Trp content results in deeper immersion of the peptide in the micelle ► Increasing Trp content dampens peptide dynamics near the water–micelle interface.

Keywords: Abbreviations; wt-TM-n-Syb; residues 75–121 of; Drosophila melanogaster; n-Synaptobrevin, biosynthetically labeled with 5-fluorotryptophan and 4-fluorophenylalanine; AA-TM-n-Syb; W89A/L90A double mutant of wt-TM-n-Syb; WW-TM-n-Syb; L90W mutant of wt-TM-n-SybNeuronal-Synaptobrevin; Membrane protein positioning; Paramagnetic contact shifts; ¹⁹; F NMR; Solvent-induced isotope shifts; Solution state NMR of membrane proteins

The lipopeptide toxins anabaenolysin A and B target biological membranes in a cholesterol-dependent manner by Linn Oftedal; Lene Myhren; Jouni Jokela; Gro Gausdal; Kaarina Sivonen; Doskeland Stein Ove Døskeland; Lars Herfindal (pp. 3000-3009).

The two novel cyanobacterial cyclic lipopeptides, anabaenolysin (Abl) A and B permeabilised mammalian cells, leading to necrotic death. Abl A was a more potent haemolysin than other known biodetergents, including digitonin, and induced discocyte–echinocyte transformation in erythrocytes. The mitochondria of the dead cells appeared intact with regard to both ultrastructure and membrane potential. Also isolated rat liver mitochondria were resistant to Abl, judged by their ultrastructure and lack of cytochrome c release. The sparing of the mitochondria could be related to the low cholesterol content of their outer membrane. In fact, a supplement of cholesterol in liposomes sensitised them to Abl. In contrast, the prokaryote-directed cyclic lipopeptide surfactin lysed preferentially non-cholesterol-containing membranes. In silico comparison of the positions of relevant functional chemical structures revealed that Abl A matched poorly with surfactin in spite of the common cyclic lipopeptide structure. Abl A and the plant-derived glycolipid digitonin had, however, predicted overlaps of functional groups, particularly in the cholesterol-binding tail of digitonin. This may suggest independent evolution of Abl and digitonin to target eukaryotic cholesterol-containing membranes. Sub-lytic concentrations of Abl A or B allowed influx of propidium iodide into cells without interfering with their long-term cell viability. The transient permeability increase allowed the influx of enough of the cyanobacterial cyclic peptide toxin nodularin to induce apoptosis. The anabaenolysins might therefore not only act solely as lysins, but also as cofactors for the internalisation of other toxins. They represent a potent alternative to digitonin to selectively disrupt cholesterol-containing biological membranes.Display Omitted► We describe the effect of two novel natural lipopeptides, anabaenolysin A and B. ► Abl A and B completely lyse the membrane of mammalian cells, but leave the mitochondria intact. ► Abl A is a more potent haemolysin than digitonin and surfactin. ► Abl A permeabilises membranes in a cholesterol-dependent manner. ► Abl A and B show a structural similarity to digitonin.

Keywords: Abbreviations; Abl; anabaenolysin; GFP; green fluorescent protein; PI; propidium iodide; SEM; scanning electron microscopy; SPE; solid phase extraction; TEM; transmission electron microscopyCyclic lipopeptide; Biodetergent; Cyanobacteria; Cholesterol; Mitochondria; Hemolysis

Interaction of local anesthetics with lipid bilayers investigated by¹H MAS NMR spectroscopy by Nicole Weizenmann; Daniel Huster; Holger A. Scheidt (pp. 3010-3018).

The membrane location of the local anesthetics (LA) lidocaine, dibucaine, tetracaine, and procaine hydrochloride as well as their influence on phospholipid bilayers were studied by³¹P and¹H magic-angle spinning (MAS) NMR spectroscopy. The³¹P NMR spectra of the LA/lipid preparations confirmed that the overall bilayer structure of the membrane remained preserved. The relation between the molecular structure of the LAs and their membrane localization and orientation was investigated quantitatively using induced chemical shifts, nuclear Overhauser enhancement spectroscopy, and paramagnetic relaxation rates. All three methods revealed an average location of the aromatic rings of all LAs in the lipid-water interface of the membrane, with small differences between the individual LAs depending on their molecular properties. While lidocaine is placed in the upper chain/glycerol region of the membrane, for dibucaine and procaine the maximum of the distribution are slightly shifted into the glycerol region. Finally for tetracaine the aromatic ring is placed closest to the aqueous phase in the glycerol/headgroup region of the membrane. The hydrophobic side chains of the LA molecules dibucaine and tetracaine were located deeper in the membrane and showed an orientation towards the hydrocarbon core. In contrast the side chains of lidocaine and procaine are oriented towards the aqueous phase.Display Omitted► Membrane location of 4 local anesthetics was studied by quantitative¹H MAS NMR. ► Aromatic rings of all local anesthetics are located in the lipid‐water interface. ► Small difference in the membrane location originate from molecular structures. ► Hydrophobic side chains of dibucaine and tetracaine are inserted into the membrane.

Keywords: Abbreviations; LA; local anesthetics; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; 5/10/16-doxyl-PC; 1-palmitoyl-2-stearoyl-(5/10/16-doxyl)-; sn; -glycero-3-phosphocholine; tempo-PC; 1,2-dipalmitoyl-; sn; -glycero-3-phospho-(tempo)choline; DMPC-; d; ₆₇; 1,2-dimyristoyl(; d; ₅₄; )-; sn; -glycero-3-phosphocholine-1,1,2,2-; d; ₄; -N,N,N-trimethyl-; d; ₉; MAS; magic angle spinning; NMR; nuclear magnetic resonance; NOESY; nuclear Overhauser enhancement spectroscopyLocal anesthetics; ¹; H NMR; Nuclear Overhauser enhancement spectroscopy; Intermolecular cross-relaxation; Chemical shift

Does cholesterol suppress the antimicrobial peptide induced disruption of lipid raft containing membranes? by Austin J. McHenry; Michele F.M. Sciacca; Jeffrey R. Brender; Ayyalusamy Ramamoorthy (pp. 3019-3024).

The activity of antimicrobial peptides has been shown to depend on the composition of the target cell membrane. The bacterial selectivity of most antimicrobial peptides has been attributed to the presence of abundant acidic phospholipids and the absence of cholesterol in bacterial membranes. The high amount of cholesterol present in eukaryotic cell membranes is thought to prevent peptide-induced membrane disruption by increasing the cohesion and stiffness of the lipid bilayer membrane. While the role of cholesterol on an antimicrobial peptide-induced membrane disrupting activity has been reported for simple, homogeneous lipid bilayer systems, it is not well understood for complex, heterogeneous lipid bilayers exhibiting phase separation (or “lipid rafts”). In this study, we show that cholesterol does not inhibit the disruption of raft-containing 1,2‐dioleoyl‐ sn‐glycero‐3‐phosphocholine:1,2‐dipalmitoyol‐ sn‐glycero‐3‐phosphocholine model membranes by four different cationic antimicrobial peptides, MSI-78, MSI-594, MSI-367 and MSI-843 which permeabilize membranes. Conversely, the presence of cholesterol effectively inhibits the disruption of non-raft containing 1,2‐dioleoyl‐ sn‐glycero‐3‐phosphocholine or 1,2‐dipalmitoyol‐ sn‐glycero‐3‐phosphocholine lipid bilayers, even for antimicrobial peptides that do not show a clear preference between the ordered gel and disordered liquid-crystalline phases. Our results show that the peptide selectivity is not only dependent on the lipid phase but also on the presence of phase separation in heterogeneous lipid systems.Display Omitted► Effect of cholesterol on antimicrobial peptide induced membrane disruption in different lipid phases was tested. ► Cholesterol had no effect on the AMP-induced membrane disruption in liquid disordered phase. ► Formation of liquid ordered phase by cholesterol strongly inhibited membrane disruption by an AMP. ► Cholesterol had no effect on the disruption of raft membranes with liquid ordered–liquid disordered coexistence.

Keywords: Abbreviations; DOPC; 1,2-dioleoyl-; sn; -glycero-3-phosphocholine; DPPC; 1,2-dipalmitoyol-; sn; -glycero-3-phosphocholine; POPC; 1-palmitoyl-2-oleoylphosphatidylcholine; SM; sphingomyelin; Chol; cholesterol; AMP; antimicrobial peptide; LUV; large unilamellar vesicle; L; _d; liquid-disordered; L; _o; liquid-ordered; L; _α; liquid-crystallineAntimicrobial peptide; Membrane selectivity; Raft domain; Phase separation; Membrane disruption

Structure analysis of the membrane-bound PhoD signal peptide of the Tat translocase shows an N-terminal amphiphilic helix by Marco J. Klein; Stephan L. Grage; Claudia Muhle-Goll; Burck Jochen Bürck; Sergii Afonin; Anne S. Ulrich (pp. 3025-3031).

Tat signal peptides provide the key signature for proteins that get exported by the bacterial twin arginine translocase. We have characterized the structure of the PhoD signal peptide from Bacillus subtilis in suitable membrane-mimicking environments. High-resolution¹³C/¹⁵N NMR analysis in detergent micelles revealed a helical stretch in the signal peptide between positions 5 and 15, in good agreement with secondary structure prediction and circular dichroism results. This helix was found to be aligned parallel to the membrane surface according to oriented circular dichroism experiments carried out with planar lipid bilayers. The N-terminal α-helix exhibits a pronounced amphiphilic character, in contrast to the general view in the literature. So far, signal sequences had been supposed to consist of a positively charged N-terminal domain, followed by an α-helical hydrophobic segment, plus a C-terminal domain carrying the peptidase cleavage site. Based on our new structural insights, we propose a model for the folding and membrane interactions of the Tat signal sequence from PhoD.Display Omitted► Structural characterization of the Tat signal peptide of PhoD in membranes ► The N-terminal domain is an amphiphilic helix that can bind to the membrane surface. ► The hydrophobic domain contains two adjacent helical segments. ► Membrane binding of the signal peptide may precede Tat translocation.

Keywords: Twin-arginine Tat protein translocation pathway; Phosphodiesterase PhoD signal sequence; Amphiphilic alpha-helix; Lipid–protein interaction; NMR structure analysis; Oriented circular dichroism

Size of the pores created by an electric pulse: Microsecond vs millisecond pulses by Gintautas Saulis; Saule Rita Saulė (pp. 3032-3039).

Here, the sizes of the pores created by square-wave electric pulses with the duration of 100μs and 2ms are compared for pulses with the amplitudes close to the threshold of electroporation. Experiments were carried out with three types of cells: mouse hepatoma MH-22A cells, Chinese hamster ovary (CHO) cells, and human erythrocytes. In the case of a short pulse (square-wave with the duration of 100μs or exponential with the time constant of 22μs), in the large portion (30–60%) of electroporated (permeable to potassium ions) cells, an electric pulse created only the pores, which were smaller than the molecule of bleomycin (molecular mass of 1450Da, r≈0.8nm) or sucrose (molecular mass of 342.3Da, radius—0.44–0.52nm). In the case of a long 2‐ms duration pulse, in almost all cells, which were electroporated, there were the pores larger than the molecules of bleomycin and/or sucrose. Kinetics of pore resealing depended on the pulse duration and was faster after the shorter pulse. After a short 100-μs duration pulse, the disappearance of the pores permeable to bleomycin was completed after 6–7min at 24–26°C, while after a long 2-ms duration pulse, this process was slower and lasted 15–20min. Thus, it can be concluded that a short 100-μs duration pulse created smaller pores than the longer 2-ms duration pulse. This could be attributed to the time inadequacy for pores to grow and expand during the pulse, in the case of short pulses.Comparison of the sizes of the pores created by square-wave electric pulses with the duration of 100μs and 2ms for pulses with the amplitudes close to the threshold of electroporation shows that a short 100μs-duration pulse creates smaller pores than the longer 2ms-duration pulse.Display Omitted► Sizes of pores created by 100μs and 2ms-duration electric pulses are compared. ► Short 100‐μs-duration pulse created smaller pores than longer 2ms-duration pulse. ► Membrane impermeability for bleomycin was restored faster after 100μs pulse.

Keywords: Electroporation; Electropermeabilization; Mouse hepatoma; CHO cells; Human erythrocytes

Aggregation behavior of ibuprofen, cholic acid and dodecylphosphocholine micelles by Priyanka Prakash; Abdallah Sayyed-Ahmad; Yong Zhou; David E. Volk; David G. Gorenstein; Elizabeth Dial; Lenard M. Lichtenberger; Alemayehu A. Gorfe (pp. 3040-3047).

Non-steroidal anti-inflammatory drugs (NSAIDs) are frequently used to treat chronic pain and inflammation. However, prolonged use of NSAIDs has been known to result in Gastrointestinal (GI) ulceration/bleeding, with a bile-mediated mechanism underlying their toxicity to the lower gut. Bile acids (BAs) and phosphatidylcholines (PCs), the major components of bile, form mixed micelles to reduce the membrane disruptive actions of monomeric BAs and simple BA micelles. NSAIDs are suspected to alter the BA/PC balance in the bile, but the molecular interactions of NSAID–BA or NSAID–BA–PC remain undetermined. In this work, we used a series of all-atom molecular dynamics simulations of cholic acid (CA), ibuprofen (IBU) and dodecylphosphocholine (DPC) mixtures to study the spontaneous aggregation of CA and IBU as well as their adsorption on a DPC micelle. We found that the size of CA–IBU mixed micelles varies with their molar ratio in a non-linear manner, and that micelles of different sizes adopt similar shapes but differ in composition and internal interactions. These observations are supported by NMR chemical shift changes, NMR ROESY crosspeaks between IBU and CA, and dynamic light scattering experiments. Smaller CA–IBU aggregates were formed in the presence of a DPC micelle due to the segregation of CA and IBU away from each other by the DPC micelle. While the larger CA–IBU aggregates arising from higher IBU concentrations might be responsible for NSAID-induced intestinal toxicity, the absence of larger CA–IBU aggregates in the presence of DPC micelles may explain the observed attenuation of NSAID toxicity by PCs.Display Omitted► Ibuprofen (an NSAID) and cholic acid (a bile acid) form mixed micelles that are toxic to the intestine. ► The size of these micelles varies non-linearly with the molar ratio of the reactants. ► Pre-formed dodecylphosphocholine micelle alters the size of the NSAID-cholic acid micelles.

Keywords: Molecular dynamics; Mixed micelle; Cluster size; NSAID; Bile acid

Detection of apoptosis through the lipid order of the outer plasma membrane leaflet by Zeinab Darwich; Andrey S. Klymchenko; Oleksandr A. Kucherak; Ludovic Richert; Mely Yves Mély (pp. 3048-3054).

Cell plasma membranes of living cells maintain their asymmetry, so that the outer leaflet presents a large quantity of sphingomyelin, which is critical for formation of ordered lipid domains. Here, a recently developed probe based on Nile Red (NR12S) was applied to monitor changes in the lipid order specifically at the outer leaflet of cell membranes. Important key features of NR12S are its ratiometric response exclusively to lipid order (liquid ordered vs. liquid disordered phase) and not to surface charge, the possibility of using it at very low concentrations (10–20nM) and the very simple staining protocol. Cholesterol extraction, oxidation and sphingomyelin hydrolysis were found to red shift the emission spectrum of NR12S, indicating a decrease in the lipid order at the outer plasma membrane leaflet. Remarkably, apoptosis induced by three different agents (actinomycin D, camptothecin, staurosporine) produced very similar spectroscopic effects, suggesting that apoptosis also significantly decreases the lipid order at this leaflet. The applicability of NR12S to detect apoptosis was further validated by fluorescence microscopy and flow cytometry, using the ratio between the blue and red parts of its emission band. Thus, for the first time, an environment-sensitive probe, sensitive to lipid order, is shown to detect apoptosis, suggesting a new concept in apoptosis sensing.Display Omitted► Outer leaflet of plasma membranes was studied by novel Nile-Red-based probe. ► Cholesterol extraction/oxidation and sphingomyelin hydrolysis decrease lipid order. ► Apoptosis induced by different agents also decreases the lipid order. ► Apoptosis and lipid order at the outer leaflet are coupled. ► Monitoring lipid order at the outer leaflet is a new concept in apoptosis detection.

Keywords: Apoptosis; Lipid order; Asymmetry of plasma membrane; Fluorescent probe; Sphingomyelin; Cholesterol

Topogenesis of heterologously expressed fragments of the human Y4 GPCR by Jacopo Marino; Eric R. Geertsma; Oliver Zerbe (pp. 3055-3063).

Fragments of large membrane proteins have the potential to facilitate structural analysis by NMR, but their folding state remains a concern. Here we determined the quality of folding upon heterologous expression for a series of N- or C-terminally truncated fragments of the human Y4 G-protein coupled receptor, amounting to six different complementation pairs. As the individual fragments lack a specific function that could be used to ascertain proper folding, we instead assessed folding on a basic level by studying their membrane topology and by comparing it to well-established structural models of GPCRs. The topology of the fragments was determined using a reporter assay based on C-terminal green fluorescent protein- or alkaline phosphatase-fusions. N-terminal fusions to Lep or Mistic were used if a periplasmic orientation of the N-terminus of the fragments was expected based on predictions. Fragments fused to Mistic expressed at comparably high levels, whereas Lep fusions were produced to a much lower extent. Though none of the fragments exclusively adopted one orientation, often the correct topology predominated. In addition, systematic analysis of the fragment series suggested that the C-terminal half of the Y4 receptor is more important for adopting the correct topology than the N-terminal part. Using the detergent dodecylphosphocholine, selected fragments were solubilized from the membrane and proved sufficiently stable to allow purification. Finally, as a first step toward reconstituting a functional receptor from two fragments, we observed a physical interaction between complementing fragments pairs upon co-expression.Display Omitted► We have prepared a systematic series of fragments of the human Y4 GPCR. ► The N terminus of the fragments is fused to Lep or Mistic, and the C terminus to either GFP or PhoA. ► GFP fluorescence and PhoA activity are measured to establish the location of the C terminus. ► All fragments assume more than one topology, but often one topology predominates. ► Two pairs of large complementary fragments are produced, purified, characterized by SEC, and shown to interact.

Keywords: GPCR; Membrane protein folding; Y-receptor; Topology screening; GFP; PhoA

Interaction of gramicidin with DPPC/DODAB bilayer fragments by Camilla A. Carvalho; Constanza Olivares-Ortega; Marco A. Soto-Arriaza; Ana M. Carmona-Ribeiro (pp. 3064-3071).

The interaction between the antimicrobial peptide gramicidin (Gr) and dipalmitoylphosphatidylcholine (DPPC)/dioctadecyldimethylammonium bromide (DODAB) 1:1 large unilamellar vesicles (LVs) or bilayer fragments (BFs) was evaluated by means of several techniques. The major methods were: 1) Gr intrinsic fluorescence and circular dichroism (CD) spectroscopy; 2) dynamic light scattering for sizing and zeta-potential analysis; 3) determination of the bilayer phase transition from extrinsic fluorescence of bilayer probes; 4) pictures of the dispersions for evaluation of coloidal stability over a range of time and NaCl concentration. For Gr in LVs, the Gr dimeric channel conformation is suggested from: 1) CD and intrinsic fluorescence spectra similar to those in trifluoroethanol (TFE); 2) KCl or glucose permeation through the LVs/Gr bilayer. For Gr in BFs, the intertwined dimeric, non-channel Gr conformation is evidenced by CD and intrinsic fluorescence spectra similar to those in ethanol. Both LVs and BFs shield Gr tryptophans against quenching by acrylamide but the Stern–Volmer quenching constant was slightly higher for Gr in BFs confirming that the peptide is more exposed to the water phase in BFs than in LVs. The DPPC/DODAB/Gr supramolecular assemblies may predict the behavior of other antimicrobial peptides in assemblies with lipids.Display Omitted► In large DPPC/DODAB 1:1vesicles, gramicidin senses a apolar medium and is in the functional channel conformation. ► KCl and glucose freely permeate through gramicidin dimeric channels in the LV bilayer. ► In DPPC/DODAB 1:1 bilayer fragments, gramicidin senses a polar medium and is intertwined. ► Interdigitation in the bilayer might have avoided Gr hydrophobic mismatch.

Keywords: Dioctadecyldimethylammonium bromide; Dipalmitoylphosphatidylcholine; Vesicle; Bilayer fragments; Gramicidin; Spectroscopic method

Imaging the membrane lytic activity of bioactive peptide latarcin 2a by Amy Won; Annamaria Ruscito; Anatoli Ianoul (pp. 3072-3080).

Latarcin 2a (ltc2a, GLFGKLIKKFGRKAISYAVKKARGKH-COOH) is a short linear antimicrobial and cytolytic peptide extracted from the venom of the Central Asian spider, Lachesana tarabaevi, with lytic activity against Gram-positive and Gram-negative bacteria, erythrocytes, and yeast at micromolar concentrations. Ltc2a adopts a helix–hinge–helix structure in membrane mimicking environment, whereas its derivative latarcin 2aG11A (ltc2aG11A, GLFGKLIKKFARKAISYAVKKARGKH-COOH), likely adopts a more rigid structure, demonstrates stronger nonspecific interaction with the zwitterionic membrane, and is potentially more toxic against eukaryotic cells. In this work, interactions of these two ltc2a derivatives with supported “raft” lipid bilayer (1,2-dioleoyl- sn-glycero-3-phosphocholin/egg sphingomyelin/cholesterol 40/40/20mol%) were studied by in situ atomic force microscopy in order to investigate the potential anticancer activity of the peptides since some breast and prostate cancer cell lines contain higher levels of cholesterol-rich lipid rafts than non-cancer cells. Both peptides induced reorganization of the raft model membrane by reducing line tension of the liquid ordered phase. Ltc2aG11A induced membrane thinning likely due to membrane interdigitation. Formation of large pores by the peptides in the bilayer was observed. Cholesterol was found to attenuate membrane disruption by the peptides. Finally, leakage assay showed that both peptides have similar membrane permeability toward various model membrane vesicles.Display Omitted► Latarcin 2a reorganizes raft model membrane. ► Mutant peptide induces membrane thinning. ► Formation of large pores by the peptides in the bilayer observed ► Cholesterol attenuates membrane disruption by the peptides.

Keywords: Supported lipid bilayer; Lipid raft; Line tension; Membrane thinning; Interdigitation; Peptide oligomerization

Voltage sensitivities and deactivation kinetics of histamine H₃ and H₄ receptors by Kristoffer Sahlholm; Johanna Nilsson; Daniel Marcellino; Kjell Fuxe; Arhem Peter Århem (pp. 3081-3089).

Agonist potency at some neurotransmitter receptors has been shown to be regulated by voltage, a mechanism which has been suggested to play a crucial role in the regulation of neurotransmitter release by inhibitory autoreceptors. Likewise, receptor deactivation rates upon agonist removal have been implicated in autoreceptor function. Using G protein-coupled potassium (GIRK) channel activation in Xenopus oocytes as readout of receptor activity, we have investigated the voltage sensitivities and signaling kinetics of the hH₃⁴⁴⁵ and hH₃³⁶⁵ isoforms of the human histamine H₃ receptor, which functions as an inhibitory auto- and heteroreceptor in the nervous system. We have also investigated both the human and the mouse homologues of the related histamine H₄ receptor, which is expressed mainly on hematopoietic cells. We found that the hH₃⁴⁴⁵ receptor is the most sensitive to voltage, whereas the hH₃³⁶⁵ and H₄ receptors are less affected. We further observed a marked difference in response deactivation kinetics between the hH₃⁴⁴⁵ and hH₃³⁶⁵ isoforms, with the hH₃³⁶⁵ isoform being five to six-fold slower than the hH₃⁴⁴⁵ receptor. Finally, using synthetic agonists, we found evidence for agonist-specific voltage sensitivity at the hH₄ receptor. The differences in voltage sensitivities and deactivation kinetics between the hH₃⁴⁴⁵, hH₃³⁶⁵, and H₄ receptors might be relevant to their respective physiological roles.Display Omitted► We study voltage sensitivity and deactivation rates at histamine H₃ and H₄ receptors. ► Histamine potency in a channel activation assay is decreased by depolarization. ► The potency decrease is more pronounced at hH₃⁴⁴⁵ than at hH₃³⁶⁵ and H₄ receptors. ► Rates of deactivation upon agonist washout differ between the histamine receptors. ► The hH₃³⁶⁵ receptor deactivates about 5 times slower than the hH₃⁴⁴⁵ receptor.

Keywords: Abbreviations; GIRK; G-protein coupled inward rectifier potassium channel; GPCR; G protein-coupled receptor; H; ₃; ⁴⁴⁵; human histamine H; ₃; ⁴⁴⁵; receptor; H; ₃; ³⁶⁵; human histamine H; ₃; ³⁶⁵; receptor; hH; ₄; human histamine H; ₄; receptor; mH; ₄; murine histamine H; ₄; receptor; RAMH; (R)-α-methylhistamine; TM; transmembrane segmentHistamine receptor; Voltage sensitivity; Voltage-clamp; Deactivation rate; Xenopus; oocyte; G protein-coupled receptor

Oligomeric state study of prokaryotic rhomboid proteases by Padmapriya Sampathkumar; Michelle W. Mak; Sarah J. Fischer-Witholt; Emmanuel Guigard; Cyril M. Kay; M. Joanne Lemieux (pp. 3090-3097).

Rhomboid peptidases (proteases) play key roles in signaling events at the membrane bilayer. Understanding the regulation of rhomboid function is crucial for insight into its mechanism of action. Here we examine the oligomeric state of three different rhomboid proteases. We subjected Haemophilus influenzae, ( hiGlpG), Escherichia coli GlpG ( ecGlpG) and Bacillus subtilis (YqgP) to sedimentation equilibrium analysis in detergent-solubilized dodecylmaltoside (DDM) solution. For hiGlpG and ecGlpG, rhomboids consisting of the core 6 transmembrane domains without and with soluble domains respectively, and YqgP, predicted to have 7 transmembrane domains with larger soluble domains at the termini, the predominant species was dimeric with low amounts of monomer and tetramers observed. To examine the effect of the membrane domain alone on oligomeric state of rhomboid, hiGlpG, the simplest form from the rhomboid class of intramembrane proteases representing the canonical rhomboid core of six transmembrane domains, was studied further. Using gel filtration and crosslinking we demonstrate that hiGlpG is dimeric and functional in DDM detergent solution. More importantly co-immunoprecipitation studies demonstrate that the dimer is present in the lipid bilayer suggesting a physiological dimer. Overall these results indicate that rhomboids form oligomers which are facilitated by the membrane domain. For hiGlpG we have shown that these oligomers exist in the lipid bilayer. This is the first detailed oligomeric state characterization of the rhomboid family of peptidases.Display Omitted► Prokaryotic rhomboids form oligomers when solubilized in detergent. ► Crosslinking and gel filtration of hiGlpG indicates a dimeric species. ► Co-IP suggests that dimers of hiGlpG form in the lipid bilayer. ► Membrane domain is the minimal unit for rhomboid oligomerization.

Keywords: Abbreviations; AU; analytical ultracentrifugation; DDM; n-dodecyl-β-; d; -maltopyranoside or dodecylmaltoside; GF; gel filtration; hi; GlpG; Haemophilius influenzae; GlpG; ec; GlpG; Escherichia coli; GlpGGlpG; Rhomboid protease; Rhomboid peptidase; Intramembrane protease; Oligomerization; Dimerization

Characterization of co-translationally formed nanodisc complexes with small multidrug transporters, proteorhodopsin and with the E. coli MraY translocase by Christian Roos; Michael Zocher; Muller Daniel Müller; Munch Daniela Münch; Tanja Schneider; Hans-Georg Sahl; Frank Scholz; Josef Wachtveitl; Yi Ma; Davide Proverbio; Erik Henrich; Dotsch Volker Dötsch; Frank Bernhard (pp. 3098-3106).

Nanodiscs (NDs) enable the analysis of membrane proteins (MP) in natural lipid bilayer environments. In combination with cell-free (CF) expression, they could be used for the co-translational insertion of MPs into defined membranes. This new approach allows the characterization of MPs without detergent contact and it could help to identify effects of particular lipids on catalytic activities. Association of MPs with different ND types, quality of the resulting MP/ND complexes as well as optimization parameters are still poorly analyzed. This study describes procedures to systematically improve CF expression protocols for the production of high quality MP/ND complexes. In order to reveal target dependent variations, the co-translational ND complex formation with the bacterial proton pump proteorhodopsin (PR), with the small multidrug resistance transporters SugE and EmrE, as well as with the Escherichia coli MraY translocase was studied. Parameters which modulate the efficiency of MP/ND complex formation have been identified and in particular effects of different lipid compositions of the ND membranes have been analyzed. Recorded force distance pattern as well as characteristic photocycle dynamics indicated the integration of functionally folded PR into NDs. Efficient complex formation of the E. coli MraY translocase was dependent on the ND size and on the lipid composition of the ND membranes. Active MraY protein could only be obtained with ND containing anionic lipids, thus providing new details for the in vitro analysis of this pharmaceutically important protein.Display Omitted► Assembly of nanodiscs with defined membrane environments ► Optimizing co-translational formation of membrane protein/nanodisc complexes ► Force distance pattern and functionality of proteorhodopsin integrated into nanodiscs ► Evaluation of lipid dependent activity of MraY translocase

Keywords: Abbreviations; AFM; atomic force microscopy; BR; bacteriorhodopsin; Cd; cardiolipin 1′,3′-bis[1,2-dimyristoyl-; sn; -glycero-3-phospho]-; sn; -glycerol; CECF; continuous exchange cell-free; CF; cell-free; DDM; n; -dodecyl-β-; d; -maltopyranoside; DHPC; 1,2-diheptanoyl-; sn; -glycero-3-phosphocholine; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphocholine; DMPG; 1,2-dimyristoyl-; sn; -glycero-3-phospho-(1′-rac-glycerol); DOPC; 1,2-dioleoyl-; sn; -glycero-3-phosphocholine; DOPE; 1,2-dioleoyl-; sn; -glycero-3-phosphoethanolamine; DOPG; 1,2-dioleoyl-; sn-; glycero-3-phospho-(1′-; rac; -glycerol); DPC; dodecyl-phosphocholine; F-D; force-distance curve; FM; feeding mix; MP; membrane protein; MSP; membrane scaffold protein; ND; nanodisc; PC; phosphocholine; PC-NBD; 1-palmitoyl-2-(12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl)-; sn; -glycero-3-phosphocholine; PE; phosphoethanolamine; PG; phosphoglycerol; PL; polar lipid extract; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phospho-(1′-; rac; -glycerol); PR; proteorhodopsin; RM; reaction mix; SDS-PAGE; sodium deodecylsulfate polyacrylamide gel electrophoreses; SEC; size exclusion chromatography; sGFP; red shifted green fluorescent protein; SMFS; single molecule force spectroscopy; SUV; small unilamellar vesicles; TL; total lipid extract; TMS; trans-membrane segments; WLC; worm-like chain fitCell-free expression; Proteorhodopsin; Nanodiscs; Lipid screening; Membrane integration; MraY translocase

Comparative study of the AT₁ receptor prodrug antagonist candesartan cilexetil with other sartans on the interactions with membrane bilayers by Charalambos Fotakis; Grigorios Megariotis; Dionysios Christodouleas; Eftichia Kritsi; Panagiotis Zoumpoulakis; Dimitrios Ntountaniotis; Maria Zervou; Constantinos Potamitis; Aden Hodzic; Georg Pabst; Michael Rappolt; Gregor Mali; Johanna Baldus; Clemens Glaubitz; Manthos G. Papadopoulos; Antreas Afantitis; Georgia Melagraki; Thomas Mavromoustakos (pp. 3107-3120).

Drug–membrane interactions of the candesartan cilexetil (TCV-116) have been studied on molecular basis by applying various complementary biophysical techniques namely differential scanning calorimetry (DSC), Raman spectroscopy, small and wide angle X-ray scattering (SAXS and WAXS), solution¹H and¹³C nuclear magnetic resonance (NMR) and solid state¹³C and³¹P (NMR) spectroscopies. In addition,³¹P cross polarization (CP) NMR broadline fitting methodology in combination with ab initio computations has been applied. Finally molecular dynamics (MD) was applied to find the low energy conformation and position of candesartan cilexetil in the bilayers. Thus, the experimental results complemented with in silico MD results provided information on the localization, orientation, and dynamic properties of TCV-116 in the lipidic environment. The effects of this prodrug have been compared with other AT₁ receptor antagonists hitherto studied. The prodrug TCV-116 as other sartans has been found to be accommodated in the polar/apolar interface of the bilayer. In particular, it anchors in the mesophase region of the lipid bilayers with the tetrazole group oriented toward the polar headgroup spanning from water interface toward the mesophase and upper segment of the hydrophobic region. In spite of their localization identity, their thermal and dynamic effects are distinct pointing out that each sartan has its own fingerprint of action in the membrane bilayer, which is determined by the parameters derived from the above mentioned biophysical techniques.Display Omitted► Drug:membrane interactions ► Biophysical methodologies (DSC, Molecular Dynamics, X-ray diffraction, NMR spectroscopy, Raman spectroscopy) ► Searching the “fingerprint” of the drug in the membrane bilayers ► AT1 antagonists and their molecular basis of action

Keywords: Candesartan cilexetil; Differential scanning calorimetry; Raman spectroscopy; Small angle X-ray scattering; Solid state NMR; Molecular dynamics

Probing ion channel activity of human islet amyloid polypeptide (amylin) by Jun Zhao; Yin Luo; Hyunbum Jang; Xiang Yu; Guanghong Wei; Ruth Nussinov; Jie Zheng (pp. 3121-3130).

Interactions of human islet amyloid polypeptide (hIAPP or amylin) with the cell membrane are correlated with the dysfunction and death of pancreatic islet β-cells in type II diabetes. Formation of receptor-independent channels by hIAPP in the membrane is regarded as one of the membrane-damaging mechanisms that induce ion homeostasis and toxicity in islet β-cells. Here, we investigate the dynamic structure, ion conductivity, and membrane interactions of hIAPP channels in the DOPC bilayer using molecular modeling and molecular dynamics simulations. We use the NMR-derived β-strand-turn-β-strand motif as a building block to computationally construct a series of annular-like hIAPP structures with different sizes and topologies. In the simulated lipid environments, the channels lose their initial continuous β-sheet network and break into oligomeric subunits, which are still loosely associated to form heterogeneous channel conformations. The channels' shapes, morphologies and dimensions are compatible with the doughnut-like images obtained by atomic force microscopy, and with those of modeled channels for Aβ, the β₂-microglobulin-derived K3 peptides, and the β-hairpin-based channels of antimicrobial peptide PG-1. Further, all channels induce directional permeability of multiple ions across the bilayers from the lower to the upper leaflet. This similarity suggests that loosely-associated β-structure motifs can be a general feature of toxic, unregulated channels. In the absence of experimental high-resolution atomic structures of hIAPP channels in the membrane, this study represents a first attempt to delineate some of the main structural features of the hIAPP channels, for a better understanding of the origin of amyloid toxicity and the development of pharmaceutical agents.Display Omitted► Structure and ion activity of hIAPP channels in DOPC bilayer are studied. ► hIAPP channels are highly selective for Cl‐ion transport. ► Multiple ion conductivity is directional from lower to upper leaflet of bilayer. ► Channel morphologies and dimensions are compatible with the experiments. ► Loosely-associated β-subunits are common structural motifs for amyloid pores.

Keywords: hIAPP; Ion channel; Directional permeability; Molecular dynamic

Ensemble and single particle fluorimetric techniques in concerted action to study the diffusion and aggregation of the glycine receptor α3 isoforms in the cell plasma membrane by Kristof Notelaers; Nick Smisdom; Susana Rocha; Daniel Janssen; Jochen C. Meier; Jean-Michel Rigo; Johan Hofkens; Marcel Ameloot (pp. 3131-3140).

The spatio-temporal membrane behavior of glycine receptors (GlyRs) is known to be of influence on receptor homeostasis and functionality. In this work, an elaborate fluorimetric strategy was applied to study the GlyR α3K and L isoforms. Previously established differential clustering, desensitization and synaptic localization of these isoforms imply that membrane behavior is crucial in determining GlyR α3 physiology. Therefore diffusion and aggregation of homomeric α3 isoform-containing GlyRs were studied in HEK 293 cells. A unique combination of multiple diffraction-limited ensemble average methods and subdiffraction single particle techniques was used in order to achieve an integrated view of receptor properties. Static measurements of aggregation were performed with image correlation spectroscopy (ICS) and, single particle based, direct stochastic optical reconstruction microscopy (dSTORM). Receptor diffusion was measured by means of raster image correlation spectroscopy (RICS), temporal image correlation spectroscopy (TICS), fluorescence recovery after photobleaching (FRAP) and single particle tracking (SPT). The results show a significant difference in diffusion coefficient and cluster size between the isoforms. This reveals a positive correlation between desensitization and diffusion and disproves the notion that receptor aggregation is a universal mechanism for accelerated desensitization. The difference in diffusion coefficient between the clustering GlyR α3L and the non-clustering GlyR α3K cannot be explained by normal diffusion. SPT measurements indicate that the α3L receptors undergo transient trapping and directed motion, while the GlyR α3K displays mild hindered diffusion. These findings are suggestive of differential molecular interaction of the isoforms after incorporation in the membrane.Display Omitted► We monitored the differential membrane behavior of glycine receptor α3 isoforms. ► Both ensemble average and single particle fluorimetric techniques were applied. ► Aggregation and diffusion were quantified using fixed and live-cell measurements. ► Evidence for molecular interaction of isoforms after membrane insertion is revealed. ► Considerations for fluorimetric probing of membrane protein behavior are reported.

Keywords: Abbreviations; 〈; r; ²; 〉; mean square displacement; ω; ₀; laser beam radius at 1/e; ²; of its maximum intensity; CLSM; confocal laser scanning microscope; D′; time-dependent diffusion coefficient; D; _1–3; diffusion coefficient derived from the time lags 1 to 3; DC; dichroic mirror; dSTORM; direct stochastic optical reconstruction microscopy; FRAP; fluorescence recovery after photobleaching; GlyR; glycine receptor; HA; hemagglutinin; HEK 293; human embryonic kidney 293; ICS; image correlation spectroscopy; MWPR; medium without phenol red; PFA; paraformaldehyde; RICS; raster image correlation spectroscopy; ROI; region of interest; SPT; single particle tracking; TICS; temporal image correlation spectroscopy; TIRFM; total internal reflection fluorescence microscope; t; _lag; time lagGlycine receptor; Alpha3 isoforms; Nanoscopy; Single particle; Ensemble average; Anomalous diffusion

Scale-independent roughness value of cell membranes studied by means of AFM technique by Palma D Antonio; Maria Lasalvia; Giuseppe Perna; Vito Capozzi (pp. 3141-3148).

The roughness of cell membrane is a very interesting indicator of cell's health state. Atomic Force Microscopy allows us to investigate the roughness of cell membrane in great detail, but the obtained roughness value is scale-dependent, i.e. it strongly depends on measurement parameters, as scanning area and step size. The scale-dependence of the roughness value can be reduced by means of data filtration techniques, that are not standardized at nanometric scale, especially as far as biological data are concerned. In this work, a new method, based on the changes of values of some roughness parameter (root mean square roughness and skewness) as a function of filtration frequencies, has been implemented to optimize data filtering procedure in the calculation of cell membrane roughness. In this way, a root mean square roughness value independent of cell shape, membrane micro-irregularities and measurement parameters can be obtained. Moreover, different filtration frequencies selected with this method allow us to discriminate different surface regimes (nominal form, waviness and roughness) belonging to the raw cell profile, each one related to different features of the cell surface.Display Omitted► The AFM technique allows us to determine cell membrane roughness value with great detail. ► The filtered roughness values are independent on the scanning scale sizes. ► Filtered roughness parameters characterize the different-size surface features.

Keywords: AFM; Cell membrane roughness; Cut-off frequency; Waviness

Differential mode of attack on membrane phospholipids by an acidic phospholipase A₂ (RVVA-PLA₂-I) from Daboia russelli venom by Debashree Saikia; Naba K. Bordoloi; Pronobesh Chattopadhyay; S. Choklingam; Siddhartha S. Ghosh; Ashis K. Mukherjee (pp. 3149-3157).

An acidic phospholipase A₂ (RVVA-PLA₂-I) purified from Daboia russelli venom demonstrated dose-dependent catalytic, mitochondrial and erythrocyte membrane damaging activities. RVVA-PLA₂-I was non‐lethal to mice at the tested dose, however, it affected the different organs of mice particularly the liver and cardiac tissues as deduced from the enzymatic activities measured in mice serum after injection of this PLA₂ enzyme. RVVA-PLA₂-I preferentially hydrolyzed phospholipids (phosphatidylcholine) of erythrocyte membrane compared to the liver mitochondrial membrane. Interestingly, RVVA-PLA₂-I failed to hydrolyze membrane phospholipids of HT-29 (colon adenocarcinoma) cells, which contain an abundance of phosphatidylcholine in its outer membrane, within 24h of incubation. The gas-chromatographic (GC) analysis of saturated/unsaturated fatty acids' release patterns from intact mitochondrial and erythrocyte membranes after the addition of RVVA-PLA₂-I showed a distinctly different result. The results are certainly a reflection of differences in the outer membrane phospholipid composition of tested membranes owing to which they are hydrolyzed by the venom PLA₂s to a different extent. The chemical modification of essential amino acids present in the active site, neutralization study with polyvalent antivenom and heat-inactivation of RVVA-PLA₂-I suggested the correlation between catalytic and membrane damaging activities of this PLA₂ enzyme. Our study advocates that the presence of a large number of PLA₂-sensitive phospholipid domains/composition, rather than only the phosphatidylcholine (PC) content of that particular membrane may determine the extent of membrane damage by a particular venom PLA₂ enzyme.An acidic PLA₂ of Daboia russelli venom preferentially hydrolyzed the outer membrane phospholipids particularly PC of erythrocytes compared to mitochondria although the latter membrane has more PC content compared to the former membrane. However, this PLA₂ failed to hydrolyze HT-29 cell membrane phospholipids. Our study advocates that the presence of a large number of PLA₂-sensitive phospholipid domains rather than only the phosphatidylcholine content of that particular membrane may determine the extent of membrane damage induced by a particular sPLA₂.GC analysis of released fatty acids from outer membrane phospholipids of intact erythrocytes post‐addition of RVVA-PLA₂-I (in the presence of Ca2⁺). A) Control, B) 15min, C) 30min and D) 60min post‐incubation of membrane with RVVA-PLA₂-I.Display Omitted► The hydrolysis of membrane phospholipids by RVV acidic PLA₂ was studied. ► Catalytic and membrane damaging activities of RVVPLA₂‐I are correlated. ► The RVVPLA₂‐I preferentially hydrolyzed PC of mitochondrial membrane. ► GC analysis of fatty acids released from different membranes demonstrated distinct results. ► Differential membrane damage suggests the presence of PLA₂-sensitive membrane domains.

Keywords: Abbreviations; pBPB; p-bromophenacyl bromide; DTT; Dithiothreitol; FA; Fatty acid; IAA; Iodoacetamide; PC; Phosphatidylcholine; PE; Phosphatidylethanolamine; PS; Phosphatidylserine; RVV; Russell's viper venom; TLCK; N-α-p-tosyl-; l; -lysine chloromethyl ketone; TPCK; Tosyl phenylalanyl chloromethyl ketoneAcidic phospholipase A; ₂; Erythrocyte; HT-29 cell; Membrane damage; Mitochondrion; Russell's viper venom PLA; ₂

Deletion of a single helix from the transmembrane domain causes large changes in membrane insertion properties and secondary structure of the bacterial conjugation protein TrwB by Ana Julia Vecino; Rosa de Lima Segura; Igor de la Arada; Fernando de la Cruz; Goni Félix M. Goñi; José L. Arrondo; Itziar Alkorta (pp. 3158-3166).

TrwB is an essential protein in the conjugative transfer of plasmid R388. The protein consists of a bulky cytosolic domain containing the catalytic site, and a small transmembrane domain (TMD). Our previous studies support the idea that the TMD plays an essential role in the activity, structure and stability of the protein. We have prepared a mutant, TrwBΔN50 that lacks one of the two α-helices in the TMD. The mutant has been studied both in detergent suspension and reconstituted in lipid vesicles. Deletion of a single helix from the TMD is enough to increase markedly the affinity of TrwB for ATP. The deletion changes the secondary structure of the cytosolic domain, whose infrared spectroscopy (IR) spectra become similar to those of the mutant TrwBΔN70 lacking the whole TMD. Interestingly, when TrwBΔN50 is reconstituted into lipid membranes, the cytosolic domain orients itself towards the vesicle interior, opposite to what happens for wild-type TrwB. In addition, we analyze the secondary structure of the TMD and TMD-lacking mutant TrwBΔN70, and found that the sum IR spectrum of the two protein fragments is different from that of the native protein, indicating the irreversibility of changes caused in TrwB by deletion of the TMD.Display Omitted► The TMD is needed for the correct folding of the cytosolic domain of TrwB. ► Deletion of a hydrophobic helix increases the affinity of TrwB for ATP. ► The orientation of TrwB into the membrane depends on the elements of the TMD.

Keywords: Abbreviations; T4CP; type IV coupling protein; TMD; transmembrane domain; IR; infrared spectroscopy; TNP-ATP; 2′(or 3′)-O-(2,4,6-trinitrophenyl) adenosine 5′‐triphosphate; PE; phosphatidylethanolamine; PG; phosphatidylglycerol; CL; cardiolipin; OG; n-octyl β-; d; -glucopyranoside; DDM; n-Dodecyl-β-; d; -maltoside; LUVs; large unilamellar vesicles; 5-IAF; 5-iodoacetamidofluorescein; AmdiS; 4-acetamido-4′-maleidylstilbene-2,2′-disulphonic acid; NEM; N-ethylmaleimide; WHH; widths at half-heightMembrane protein; Transmembrane domain; Membrane protein reconstitution; Membrane protein orientation; Infrared spectroscopy; Conjugative coupling protein

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BBA - Biomembranes (v.1818, #12)

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BBA - Biomembranes (v.1818, #12)