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BBA - Biomembranes (v.1828, #3)

Editorial Board (pp. i).

Differential effect of sterols on dipole potential in hippocampal membranes: Implications for receptor function by Pushpendra Singh; Sourav Haldar; Amitabha Chattopadhyay (pp. 917-923).

Dipole potential is the potential difference within the membrane bilayer, which originates due to the nonrandom arrangement of lipid dipoles and water molecules at the membrane interface. In this work, we have explored the possible correlation between functional modulation of a G protein-coupled receptor (the serotonin_1A receptor) and membrane dipole potential, under conditions of altered membrane sterol composition. We have previously shown that the ligand binding activity of the hippocampal serotonin_1A receptor is reduced upon cholesterol depletion and could be restored upon replenishment with cholesterol. Interestingly, when the replenishment was carried out with an immediate biosynthetic precursor of cholesterol (7-DHC), differing with cholesterol merely in a double bond, the ligand binding activity of the receptor was not restored. In order to understand the mechanistic framework of receptor–cholesterol interaction, we carried out dipole potential measurements of hippocampal membranes under these conditions, by the dual wavelength ratiometric approach using an electrochromic probe (di-8-ANEPPS). We show here that dipole potential of hippocampal membranes is reduced upon progressive depletion of cholesterol and is restored upon replenishment with cholesterol, but not with 7-DHC. Our results show that the recovery of ligand binding activity of the serotonin_1A receptor upon replenishment with cholesterol (but not with 7-DHC) could be related to the differential ability of these closely related sterols to modulate membrane dipole potential. We conclude that subtle changes in membrane dipole potential could be crucial in understanding the complex interplay between membrane lipids and proteins in the cellular milieu.Display Omitted► Dipole potential of hippocampal membranes is reduced upon cholesterol depletion ► Replenishment with cholesterol restores dipole potential in hippocampal membranes ► 7-DHC replenishment does not restore dipole potential in hippocampal membranes ► Unlike cholesterol, 7-DHC does not support function of the serotonin_1A receptor ► Membrane dipole potential could play crucial role in the lipid–protein interaction.

Keywords: Abbreviations; 7-DHC; 7-dehydrocholesterol; 8-OH-DPAT; 8-hydroxy-2(di-; N; -propylamino)tetralin; BCA; bicinchoninic acid; di-8-ANEPPS; 4-(2-(6-(dioctylamino)-2-naphthalenyl)ethenyl)-1-(3-sulfopropyl)-pyridinium inner salt; DMPC; dimyristoyl-; sn; -glycero-3-phosphocholine; DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phosphocholine; GPCR; G protein-coupled receptor; LUV; large unilamellar vesicle; MβCD; methyl-β-cyclodextrin; PMSF; phenylmethylsulfonyl fluoride; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; SLOS; Smith–Lemli–Opitz Syndrome; TLC; thin layer chromatography; Tris; tris; -(hydroxymethyl)aminomethaneDipole potential; Cholesterol; 7-DHC; Serotonin; _1A; receptor; Ligand binding; di-8-ANEPPS

Freely turning over palmitate in erythrocyte membrane proteins is not responsible for the anchoring of lipid rafts to the spectrin skeleton: A study with bio-orthogonal chemical probes by Annarita Ciana; Cesare Achilli; Rami N. Hannoush; Angela Risso; Cesare Balduini; Giampaolo Minetti (pp. 924-931).

Erythrocyte lipid rafts are anchored to the underlying spectrin membrane skeleton [A. Ciana, C. Achilli, C. Balduini, G. Minetti, On the association of lipid rafts to the spectrin skeleton in human erythrocytes, Biochim. Biophys. Acta 1808 (2011) 183–190]. The nature of this linkage and the molecules involved are poorly understood. The interaction is sensitive to the increase in pH and ionic strength induced by carbonate. Given the role of palmitoylation in modulating the partitioning of certain proteins between various sub-cellular compartments and the plasma membrane, we asked whether palmitoylation of p55, a peripheral protein located at the junctional complex between spectrin-actin-protein 4.1 that anchors the membrane skeleton to the lipid bilayer via the transmembrane protein glycophorin C, could contribute to the anchoring of lipid rafts to the membrane skeleton. We adopted a new, non-radioactive method for studying protein palmitoylation, based on bio-orthogonal chemical analogues of fatty acids, containing an omega-alkynyl group, to metabolically label cell proteins, which are then revealed by a “click chemistry” reaction of the alkynyl moiety with an azide-containing reporter tag. We show that the membrane localization and palmitoylation levels of p55 did not change after carbonate treatment. 2-bromopalmitate and cerulenin, two known palmitoylation inhibitors, completely inhibited p55 palmitoylation, and protein palmitoyl thioesterase-1 (PPT1) reduced it, without affecting the association between lipid rafts and membrane-skeleton, indicating, on the one hand, that p55 palmitoylation is enzymatic, and, on the other, that it is not involved in the modulation of the linkage of lipid rafts to the membrane-skeleton.Display Omitted► In erythrocytes, lipid rafts are anchored to the spectrin skeleton in a carbonate-sensitive manner. ► The palmitoylated protein p55 could contribute to this anchoring via its palmitate. ► p55 palmitoylation was studied with an ω-alkynyl analogue of palmitic acid and click chemistry. ► Carbonate does not depalmitoylate p55 but releases lipid rafts from membrane-skeleton. ► 2-bromopalmitate and cerulenin inhibit p55 palmitoylation without affecting lipid raft anchoring.

Keywords: Abbreviations; Alk-C16; ω-alkynyl palmitic acid; 2BP; 2-bromopalmitic acid; DFP; di-isopropyl fluoro phosphate; DRM; detergent-resistant membranes; GPC; glycophorin C; HRP; horseradish peroxidase; PPT1; protein palmitoyl thioesterase 1; TX100; Triton X-100Fatty acylation; Detergent-resistant membrane; 2-bromopalmitate; Cerulenin; Click chemistry; Omega-alkynyl

Sterol affinity for phospholipid bilayers is influenced by hydrophobic matching between lipids and transmembrane peptides by Ijas H. Kristian Ijäs; Lonnfors Max Lönnfors; Thomas K.M. Nyholm (pp. 932-937).

Lipid self-organization is believed to be essential for shaping the lateral structure of membranes, but it is becoming increasingly clear that also membrane proteins can be involved in the maintenance of membrane architecture. Cholesterol is thought to be important for the lateral organization of eukaryotic cell membranes and has also been implicated to take part in the sorting of cellular transmembrane proteins. Hence, a good starting point for studying the influence of lipid–protein interactions on membrane trafficking is to find out how transmembrane proteins influence the lateral sorting of cholesterol in phospholipid bilayers. By measuring equilibrium partitioning of the fluorescent cholesterol analog cholestatrienol between large unilamellar vesicles and methyl-β-cyclodextrin the effect of hydrophobic matching on the affinity of sterols for phospholipid bilayers was determined. Sterol partitioning was measured in 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers with and without WALP19, WALP23 or WALP27 peptides. The results showed that the affinity of the sterol for the bilayers was affected by hydrophobic matching. An increasing positive hydrophobic mismatch led to stronger sterol binding to the bilayers (except in extreme situations), and a large negative hydrophobic mismatch decreased the affinity of the sterol for the bilayer. In addition, peptide insertion into the phospholipid bilayers was observed to depend on hydrophobic matching. In conclusion, the results showed that hydrophobic matching can affect lipid–protein interactions in a way that may facilitate the formation of lateral domains in cell membranes. This could be of importance in membrane trafficking.Display Omitted► The sterol affinity for phospholipid bilayers is affected by transmembrane peptides. ► Hydrophobic matching influences how the sterol affinity is affected by peptides. ► Peptides can both decrease and increase the sterol's affinity for the bilayer. ► Hydrophobic matching could control lateral organization in cellular membranes.

Keywords: Abbreviations; CTL; cholesta-5,7,9 (11)-trien-3-beta-ol; DPH-PC; 2-(3-(diphenylhexatrienyl)propanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine; K; _X; the molar fraction partitioning coefficient; K; _R; the relative partitioning coefficient; DLPC; 1,2-dilauroyl-sn-glycero-3-phosphocholine; DMPC; 1,2-dimyristoyl-sn-glycero-3-phosphocholine; DPPC; 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; mβCD; methyl-β-cyclodextrin; LUV; large unilamellar vesicleModel membrane; Fluorescence spectroscopy; Protein–lipid interaction; Cholesterol; Membrane trafficking; Protein sorting

Lipid modification gives rise to two distinct Haloferax volcanii S-layer glycoprotein populations by Lina Kandiba; Ziqiang Guan; Jerry Eichler (pp. 938-943).

The S-layer glycoprotein is the sole component of the protein shell surrounding Haloferax volcanii cells. The deduced amino acid sequence of the S-layer glycoprotein predicts the presence of a C-terminal membrane-spanning domain. However, several earlier observations, including the ability of EDTA to selectively solubilize the protein, are inconsistent with the presence of a trans-membrane sequence. In the present report, sequential solubilization of the S-layer glycoprotein by EDTA and then with detergent revealed the existence of two distinct populations of the S-layer glycoprotein. Whereas both S-layer glycoprotein populations underwent signal peptide cleavage and N-glycosylation, base hydrolysis followed by mass spectrometry revealed that a lipid, likely archaetidic acid, modified only the EDTA-solubilized version of the protein. These observations are consistent with the S-layer glycoprotein being initially synthesized as an integral membrane protein and subsequently undergoing a processing event in which the extracellular portion of the protein is separated from the membrane-spanning domain and transferred to a waiting lipid moiety.Display Omitted► The S-layer glycoprotein solely comprises the Haloferax volcanii surface layer ► Sequential EDTA and Triton X-100 treatment expose two distinct forms of the protein ► Both S-layer glycoprotein forms undergo signal peptide cleavage and N-glycosylation ► Only EDTA-solubilized S-layer glycoprotein is lipid-modified.

Keywords: Abbreviations; liquid chromatography-electrospray ionization mass spectrometry; LC-ESI/MS; methanol; MeOH; surface layer; S-layer; trichloroacetic acid; TCAArchaea; Haloferax volcanii; Lipid modification; Membrane protein; S-layer glycoprotein

Design and characterization of novel antimicrobial peptides, R-BP100 and RW-BP100, with activity against Gram-negative and Gram-positive bacteria by Inês M. Torcato; Yen-Hua Huang; Henri G. Franquelim; Diana Gaspar; David J. Craik; Miguel A.R.B. Castanho; Sónia Troeira Henriques (pp. 944-955).

BP100 is a short cationic antimicrobial peptide with a mechanism of action dependent on peptide–lipid interactions and microbial surface charge neutralization. Although active against Gram-negative bacteria, BP100 is inactive against Gram-positive bacteria. In this study we report two newly designed BP100 analogues, RW-BP100 and R-BP100 that have the Tyr residue replaced with a Trp and/or the Lys residues replaced with an Arg. The new analogues in addition to being active against Gram-negative bacteria, possess activity against all tested Gram-positive bacteria. Mechanistic studies using atomic force microscopy, surface plasmon resonance and fluorescence methodologies reveal that the antibacterial efficiency follows the affinity for bacterial membrane. The studies suggest that the activity of BP100 and its analogues against Gram-negative bacteria is mainly driven by electrostatic interactions with the lipopolysaccharide layer and is followed by binding to and disruption of the inner membrane, whereas activity against Gram-positive bacteria, in addition to electrostatic attraction to the exposed lipoteichoic acids, requires an ability to more deeply insert in the membrane environment, which is favoured with Arg residues and is facilitated in the presence of a Trp residue. Knowledge on the mechanism of action of these antimicrobial peptides provides information that assists in the design of antimicrobials with higher efficacy and broader spectra of action, but also on the design of peptides with higher specificity if required.Display Omitted► Two novel antimicrobial peptides (AMPs) active against Gram− and Gram+ bacteria ► Arg instead of Lys residues improve AMP activity against Gram+ bacteria. ► AMP mode of action against Gram+ requires electrostatic binding but also deeply insertion in the membrane. ► Lys vs Arg residues relevant for AMP specificity vs broad spectra of action

Keywords: Abbreviations; H-bond; hydrogen bond; AMP; antimicrobial peptide; AFM; atomic force microscopy; RP-HPLC; reversed phase high-performance liquid chromatography; MBC; minimal bactericidal concentration; MIC; minimal inhibitory concentration; E. coli; Escherichia coli; S. aureus; Staphylococcus aureus; K. pneumoniae; Klebsiella pneumonia; P. aeroginosa; Pseudomonas aeroginosa; S. pneumonia; Streptococcus pneumonia; E. faecium; Enterococcus faecium; ATCC; American type culture collection; MHB; Mueller Hinton broth; OD; optical density; PBS; phosphate buffered saline; NaCl; sodium chloride; S; _rms; bacterial shape; R; _rms; cell surface roughness; RBC; red blood cell; HC; ₅₀; peptide concentration needed to achieve 50% of haemolysis; IC; ₅₀; peptide concentration needed to achieve 50% of cell death; REES; red-edge excitation shift; HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; LUV; large unilamellar vesicles; POPC; 1-palmitoyl-2-Oleoyl-; sn; -Glycero-3-Phosphocholine; POPG; 1-palmitoyl-2-Oleoyl-; sn; -Glycero-3-Phosphoglycerol; 5/16-NS; 5/16- doxyl stearic acid; CD; circular dichroism; di-8-ANEPPS; 4-[2-[6-(dioctylamino)-2-naphthalenyl]ethenyl]-1-(3-sulfopropyl); PBMC; peripheral blood mononuclear cell; CF; carboxyfluorescein; LC; ₅₀; peptide concentration needed to induce 50% of leakage; T; _1/2; time necessary to achieve 50% of leakage; LPS; lipopolysaccharide; LTA; lipoteichoic acidAntimicrobial peptide; Model membrane; Peptide–membrane interaction; Atomic force microscopy; Trp/Tyr fluorescence

Stomatin interacts with GLUT1/SLC2A1, band 3/SLC4A1, and aquaporin-1 in human erythrocyte membrane domains by Stefanie Rungaldier; Walter Oberwagner; Ulrich Salzer; Edina Csaszar; Rainer Prohaska (pp. 956-966).

The widely expressed, homo-oligomeric, lipid raft-associated, monotopic integral membrane protein stomatin and its homologues are known to interact with and modulate various ion channels and transporters. Stomatin is a major protein of the human erythrocyte membrane, where it associates with and modifies the glucose transporter GLUT1; however, previous attempts to purify hetero-oligomeric stomatin complexes for biochemical analysis have failed. Because lateral interactions of membrane proteins may be short-lived and unstable, we have used in situ chemical cross-linking of erythrocyte membranes to fix the stomatin complexes for subsequent purification by immunoaffinity chromatography. To further enrich stomatin, we prepared detergent-resistant membranes either before or after cross-linking. Mass spectrometry of the isolated, high molecular, cross-linked stomatin complexes revealed the major interaction partners as glucose transporter-1 (GLUT1), anion exchanger (band 3), and water channel (aquaporin-1). Moreover, ferroportin-1 (SLC40A1), urea transporter-1 (SLC14A1), nucleoside transporter (SLC29A1), the calcium-pump (Ca-ATPase-4), CD47, and flotillins were identified as stomatin-interacting proteins. These findings are in line with the hypothesis that stomatin plays a role as membrane-bound scaffolding protein modulating transport proteins.Display Omitted► Stomatin is chemically cross-linked in erythrocyte membranes ► Stomatin complexes are immunoaffinity-purified and identified by mass spectrometry ► Major cross-linked partners are GLUT1 and band 3 ► Stomatin is cross-linked in detergent-resistant membranes ► New interaction partners include flotillins and various transporters/channels, particularly aquaporin-1.

Keywords: Abbreviations; ASIC; acid-sensing ion channel; AvTIC; average total ion current; DRM; detergent-resistant membrane; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; LC-MS/MS; liquid chromatography tandem MS; MS; mass spectrometry; PMSF; phenylmethylsulfonyl fluoride; TNE; Tris/NaCl/EDTA buffer; TNET; Tris/NaCl/EDTA/Triton X-100 bufferIntegral membrane proteins; Lipid rafts; Chemical cross-linking; Protein–protein interaction

Interactions of Thellungiella salsuginea dehydrins TsDHN-1 and TsDHN-2 with membranes at cold and ambient temperatures—Surface morphology and single-molecule force measurements show phase separation, and reveal tertiary and quaternary associations by Luna N. Rahman; Fraser McKay; Maximiliano Giuliani; Amanda Quirk; Barbara A. Moffatt; George Harauz; John R. Dutcher (pp. 967-980).

Dehydrins (group 2 late embryogenesis abundant proteins) are intrinsically-disordered proteins that are expressed in plants experiencing extreme environmental conditions such as drought or low temperature. Their roles include stabilizing cellular proteins and membranes, and sequestering metal ions. Here, we investigate the membrane interactions of the acidic dehydrin TsDHN-1 and the basic dehydrin TsDHN-2 derived from the crucifer Thellungiella salsuginea that thrives in the Canadian sub-Arctic. We show using compression studies with a Langmuir–Blodgett trough that both dehydrins can stabilize lipid monolayers with a lipid composition mimicking the composition of the plant outer mitochondrial membrane, which had previously been shown to induce ordered secondary structures (disorder-to-order transitions) in the proteins. Ellipsometry of the monolayers during compression showed an increase in monolayer thickness upon introducing TsDHN-1 (acidic) at 4°C and TsDHN-2 (basic) at room temperature. Atomic force microscopy of supported lipid bilayers showed temperature-dependent phase transitions and domain formation induced by the proteins. These results support the conjecture that acidic dehydrins interact with and potentially stabilize plant outer mitochondrial membranes in conditions of cold stress. Single-molecule force spectroscopy of both proteins pulled from supported lipid bilayers indicated the induced formation of tertiary conformations in both proteins, and potentially a dimeric association for TsDHN-2.Display Omitted► Thellungiella salsuginea dehydrins TsDHN-1 and TsDHN-2 are expressed during cold stress. ► Both dehydrins associate with lipid monolayers and bilayers, and cause domain formation. ► Single molecule force microscopy shows both proteins to undergo tertiary compaction on membranes. ► TsDHN-2 also undergoes potential dimerization. ► Results support that these dehydrins stabilize plant membranes during cold stress.

Keywords: Abbreviations; AFM; atomic force microscopy; ATR; attenuated total reflectance; Chol; cholesterol; FTIR; Fourier transform infrared; LEA; late embryogenesis abundant; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PG; phosphatidylglycerol; PS; phosphatidylserine; SMFS; single-molecule force spectroscopy; SUMO; small ubiquitin modifier; TsDHN-1; acidic; Thellungiella salsuginea; dehydrin 1; TsDHN-2; basic; Thellungiella salsuginea; dehydrin 2Dehydrin; Intrinsically-disordered protein; Cold and drought tolerance; Langmuir–Blodgett monolayers; Supported lipid bilayer; Atomic force microscopy

Primary pathways of intracellular Ca2⁺ mobilization by nanosecond pulsed electric field by Iurii Semenov; Shu Xiao; Andrei G. Pakhomov (pp. 981-989).

Permeabilization of cell membranous structures by nanosecond pulsed electric field (nsPEF) triggers transient rise of cytosolic Ca2⁺ concentration ([Ca2⁺]_i), which determines multifarious downstream effects. By using fast ratiometric Ca2⁺ imaging with Fura-2, we quantified the external Ca2⁺ uptake, compared it with Ca2⁺ release from the endoplasmic reticulum (ER), and analyzed the interplay of these processes. We utilized CHO cells which lack voltage-gated Ca2⁺ channels, so that the nsPEF-induced [Ca2⁺]_i changes could be attributed primarily to electroporation. We found that a single 60-ns pulse caused fast [Ca2⁺]_i increase by Ca2⁺ influx from the outside and Ca2⁺ efflux from the ER, with the E-field thresholds of about 9 and 19kV/cm, respectively. Above these thresholds, the amplitude of [Ca2⁺]_i response increased linearly by 8–10nM per 1kV/cm until a critical level between 200 and 300nM of [Ca2⁺]_i was reached. If the critical level was reached, the nsPEF-induced Ca2⁺ signal was amplified up to 3000nM by engaging the physiological mechanism of Ca2⁺-induced Ca2⁺-release (CICR). The amplification was prevented by depleting Ca2⁺ from the ER store with 100nM thapsigargin, as well as by blocking the ER inositol-1,4,5-trisphosphate receptors (IP₃R) with 50μM of 2-aminoethoxydiphenyl borate (2-APB). Mobilization of [Ca2⁺]_i by nsPEF mimicked native Ca2⁺ signaling, but without preceding activation of plasma membrane receptors or channels. NsPEF stimulation may serve as a unique method to mobilize [Ca2⁺]_i and activate downstream cascades while bypassing the plasma membrane receptors.Display Omitted► Effects of 60ns PEF on [Ca2⁺]_i were studied by using fast measurements with Fura-2. ► NsPEF induced Ca2⁺ uptake via plasma membrane and Ca2⁺ efflux from ER. ► NsPEF of higher amplitudes triggered amplification of Ca2⁺ rise via CICR mechanism. ► Blocking of IP₃R abolished the amplification of Ca2⁺ response to nsPEF.

Keywords: Abbreviations; 2-APB; 2-aminoethoxydiphenyl borate; CICR; Ca; 2; ⁺; -induced Ca; 2; ⁺; -release; [Ca; 2; ⁺; ]; _i; intracellular concentration of free Ca; 2; ⁺; ions; [Ca; 2; ⁺; ]; _o; extracellular concentration of free Ca; 2; ⁺; ions; DMSO; dimethylsolfoxide; EGTA; ethylene glycol tetraacetic acid; ER; endoplasmic reticulum; FBS; fetal bovine serum; IP; ₃; R; inositol-1,4,5-trisphosphate receptor; K; _d; dissociation constant; NCX; sodium–calcium exchanger; nsPEF; nanosecond pulsed electric field; PMCA; plasma membrane Ca; 2; ⁺; ATPase; RyR; ryanodine receptor; SERCA; sarco/endoplasmic reticulum Ca; 2; ⁺; -ATPase; TG; thapsigarginNanosecond pulse; Electroporation; Calcium signaling; Membrane permeabilization

Effect of inserting charged peptide at NH₂-terminal on N-type inactivation of Kv1.4 channel by Zhuo Fan; Zhenggang Zhang; Mingyu Fu; Zhi Qi; Zhongju Xiao (pp. 990-996).

Rapid inactivation of voltage-gated potassium channel plays an important role in shaping the electrical signaling in neurons and other excitable cells. N-type (“ball and chain”) inactivation, as the most extensively studied inactivation model, is assumed to be the inactivation mechanism of Kv1.4 channel. The inactivation ball inactivates the channel by interacting with the hydrophobic wall of inner pore and occluding it. Recently, we have proved that the electrostatic interaction between two charged segments in the NH₂-termainal plays an important role through promoting the inactivation process of the Kv1.4 channel. This study investigates the effect of inserting negatively or positively charged short peptides at NH₂-terminal on the inactivation of Kv1.4 channel. The results that inserting negatively-charged peptide (either myc or D-peptide) at different sites of NH₂-terminal, deceleraes inactivation process of Kv1.4 channel to a different extent with inserting site changing and that the mutant Kv1.4-D50 exhibits a more slower inactivation rate than Kv1.4-K50 further identified the role of electrostatic interactions in the “ball and chain” inactivation mechanism.Display Omitted► Effect of inserting negatively-charged c-myc tag at NH₂-terminal of Kv1.4. ► Effect of inserting negatively-charged D-peptide at the same sites. ► D-peptide decelerated the inactivation to a greater extent than myc-peptide. ► Effect of inserting positively-charged K-peptide at NH₂-terminal of Kv1.4. ► The voltage-dependence of inactivation or activation is not changed.

Keywords: Kv1.4 channel; N-type inactivation; “ball and chain” model; Electrostatic interaction; Voltage-gated ion channel; Hydrophobic interaction

Identification of important charged residues for alkali cation exchange or pH regulation of NhaH, a Na⁺/H⁺ antiporter of Halobacillus dabanensis by Juquan Jiang; Lei Wang; Yajie Zou; Weidong Lu; Baisuo Zhao; Bo Zhang; Susheng Yang; Lifu Yang (pp. 997-1003).

NhaH is a novel Na⁺/H⁺ antiporter identified from the moderate halophile Halobacillus dabanensis. In this study, six conserved charged residues located in the putative trans membrane segments (TMS) including TMSV, TMSVI, TMSVIII and TMSXI of NhaH as well as two His residues in Loop III were replaced by site-directed mutagenesis for the identification of their potential roles in the antiport activity and pH regulation. Substitutions D137A, D166A and R325A caused a complete loss of Na⁺(Li⁺)/H⁺ antiport activity, revealing that D137, D166 and R325 are indispensable for the antiport activity. Substitution D137E led to a significant increase of the apparent Km values for Na⁺ and Li⁺ without affecting the changes of pH profile, confirming that D137 plays vital roles in alkali cation binding/translocation. Substitution D166E resulted in not only a significant increase of the apparent Km values for Na⁺ and Li⁺ but also an alkaline shift of pH profile, suggesting that D166 is involved in alkali cation binding/translocation as well as H⁺ binding or pH regulation. Substitutions E161N, D224A and D224E caused a significant increase of Km for Na⁺ and Li⁺, indicating that E161 and D224 partly contribute to alkali cation binding/translocation. Substitution E229K caused an over 50% elevation of the apparent Km for Li⁺, without affecting that for Na⁺, suggesting that E229 may be mainly responsible for Li⁺ binding/translocation. Substitutions H87A and H88A resulted in an acidic shift of pH profile without an effect on Km for Na⁺ and Li⁺, indicating that H87 and H88 are involved in H⁺ binding or pH regulation.Display Omitted► Relationships between eight important charged residues and the function or pH regulation of NhaH were studied. ► D137, D166 and R325 are indispensable for the Na⁺(Li⁺)/H⁺ antiport activity. ► E161 and D224 partly contribute to the alkali cation binding/translocation. ► E229 may be mainly responsible for Li⁺ binding/translocation. ► H87, H88 and D166 are involved in H⁺ binding or pH regulation.

Keywords: Halobacillus dabanensis; NhaH; Antiporter; Site-directed mutagenesis; Alkali cation translocation; pH regulation

Selective antimicrobial activity and mode of action of adepantins, glycine-rich peptide antibiotics based on anuran antimicrobial peptide sequences by Ilic Nada Ilić; Novkovic Mario Novković; Filomena Guida; Daniela Xhindoli; Monica Benincasa; Alessandro Tossi; Juretic Davor Juretić (pp. 1004-1012).

A challenge when designing membrane-active peptide antibiotics with therapeutic potential is how to ensure a useful antibacterial activity whilst avoiding unacceptable cytotoxicity for host cells. Understanding their mode of interaction with membranes and the reasons underlying their ability to distinguish between bacterial and eukaryotic cytoplasmic cells is crucial for any rational attempt to improve this selectivity. We have approached this problem by analysing natural helical antimicrobial peptides of anuran origin, using a structure–activity database to determine an antimicrobial selectivity index (SI) relating the minimal inhibitory concentration against Escherichia coli to the haemolytic activity (SI=HC₅₀/MIC). A parameter that correlated strongly with SI, derived from the lengthwise asymmetry of the peptides' hydrophobicity (sequence moment), was then used in the “Designer” algorithm to propose novel, highly selective peptides. Amongst these are the ‘adepantins’, peptides rich in glycines and lysines that are highly selective for Gram-negative bacteria, have an exceptionally low haemolytic activity, and are less than 50% homologous to any other natural or synthetic antimicrobial peptide. In particular, they showed a very high SI for E. coli (up to 400) whilst maintaining an antimicrobial activity in the 0.5–4μM range. Experiments with monomeric, dimeric and fluorescently labelled versions of the adepantins, using different bacterial strains, host cells and model membrane systems provided insight into their mechanism of action.Display Omitted► Algorithm was trained on anuran AMPs with known haemolytic/antimicrobial activities. ► We used it to design AMPs highly selective for Gram-negative bacteria. ► Monomeric and dimeric analogues were prepared to study the mode of action. ► The peptides act on the bacterial membrane in a highly selective manner.

Keywords: Abbreviations; AMP; antimicrobial peptide; BODIPY; boron-dipyrromethene; Gal-ONp; o-nitrophenyl-β-D-galactopyranoside; HC; ₅₀; concentration for 50% haemolysis; IC; ₅₀; concentration for 50% of growth inhibition; LUVs; large unilamellar vesicles; MIC; minimum inhibitory concentration; MBC; minimal bactericidal concentration; PI; propidium iodide; TFE; trifluoroethanol; SI; antimicrobial selectivity indexAntimicrobial peptide; Helical peptide; Membranolytic; Selectivity index

Membrane thickness and the mechanism of action of the short peptaibol trichogin GA IV by S. Bobone; Y. Gerelli; M. De Zotti; G. Bocchinfuso; A. Farrotti; B. Orioni; F. Sebastiani; E. Latter; J. Penfold; R. Senesi; F. Formaggio; A. Palleschi; C. Toniolo; G. Fragneto; L. Stella (pp. 1013-1024).

Trichogin GA IV (GAIV) is an antimicrobial peptide of the peptaibol family, like the extensively studied alamethicin (Alm). GAIV acts by perturbing membrane permeability. Previous data have shown that pore formation is related to GAIV aggregation and insertion in the hydrophobic core of the membrane. This behavior is similar to that of Alm and in agreement with a barrel-stave mechanism, in which transmembrane oriented peptides aggregate to form a channel. However, while the 19-amino acid long Alm has a length comparable to the membrane thickness, GAIV comprises only 10 amino acids, and its helix is about half the normal bilayer thickness. Here, we report the results of neutron reflectivity measurements, showing that GAIV inserts in the hydrophobic region of the membrane, causing a significant thinning of the bilayer. Molecular dynamics simulations of GAIV/membrane systems were also performed. For these studies we developed a novel approach for constructing the initial configuration, by embedding the short peptide in the hydrophobic core of the bilayer. These calculations indicated that in the transmembrane orientation GAIV interacts strongly with the polar phospholipid headgroups, drawing them towards its N- and C-termini, inducing membrane thinning and becoming able to span the bilayer. Finally, vesicle leakage experiments demonstrated that GAIV activity is significantly higher with thinner membranes, becoming similar to that of Alm when the bilayer thickness is comparable to its size. Overall, these data indicate that a barrel-stave mechanism of pore formation might be possible for GAIV and for similarly short peptaibols despite their relatively small size.Display Omitted► The short peptaibol trichogin GAIV inserts in the core of the membrane. ► Once inserted, it causes a significant thinning of the bilayer. ► According to simulations, this is due to its interaction with lipid headgroups. ► Its activity increases dramatically in thinner membranes. ► It might form barrel-stave channels, like the longer peptaibol alamethicin.

Keywords: Abbreviations; HFIP; 1,1,1,3,3,3-hexafluoroisopropanol; Aib; α-aminoisobutyric acid; Alm; alamethicin; AMP; antimicrobial peptide; CF; carboxyfluorescein; DIEA; N,N-diisopropylethylamine; DMF; N,N-dimethylformamide; EDC; N-ethyl, N′-[3-(dimethylamino)propyl]carbodiimide; Fmoc; fluorenyl-9-methyloxycarbonyl; GAIV; trichogin GAIV; HATU; O-(7-azabenzotriazol-1-yl)-1,1,3,3 tetramethyluronium hexafluorophosphate; HOAt; 7-aza-1-hydroxy-benzotriazole; Lol; leucinol; MD; molecular dynamics; n; Oct; n; -octanoyl; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; SLD; scattering length density; SMH; Shai–Matsuzaki–HuangMembrane thickness; Molecular dynamics simulations; Neutron reflectivity; Peptaibiotics; Peptide embedding in a bilayer; Transmembrane peptides

Growth kinetics of lipid-based nanodiscs to unilamellar vesicles—A time-resolved small angle neutron scattering (SANS) study by Suanne Mahabir; Darcy Small; Ming Li; Wankei Wan; Kucerka Norbert Kučerka; Kenneth Littrell; John Katsaras; Mu-Ping Nieh (pp. 1025-1035).

Mixtures of dimyristoyl-phosphatidylcholine (DMPC), dimyristoyl-phosphatidylglycerol (DMPG) and dihexanoyl-phosphatidylcholine (DHPC) in aqueous solutions spontaneously form monodisperse, bilayered nanodiscs (also known as “bicelles”) at or below the melting transition temperature of DMPC (T _M ~23°C). In dilute systems above the main transition temperatureT _M of DMPC, bicelles coalesce (increasing their diameter) and eventually self-fold into unilamellar vesicles (ULVs). Time-resolved small angle neutron scattering was used to study the growth kinetics of nanodiscs below and equal toT _M over a period of hours as a function of temperature at two lipid concentrations in presence or absence of NaCl salt. Bicelles seem to undergo a sudden initial growth phase with increased temperature, which is then followed by a slower reaction-limited growth phase that depends on ionic strength, lipid concentration and temperature. The bicelle interaction energy was derived from the colloidal theory of Derjaguin and Landau, and Verwey and Overbeek (DLVO). While the calculated total energy between discs is attractive and proportional to their growth rate, a more detailed mechanism is proposed to describe the mechanism of disc coalescence. After annealing at low temperature (low- T), samples were heated to 50°C in order to promote the formation of ULVs. Although the low- T annealing of samples has only a marginal effect on the mean size of end-state ULVs, it does affect their polydispersity, which increases with increased T, presumably driven by the entropy of the system.Display Omitted► The growth mechanism of lipid-based nanodiscs is examined by time-resolved SANS. ► DLVO theory is used to partially describe the interparticle interaction. ► The growth of the nanodiscs is a reaction-limited coalescence process. ► A nanodisc-to-vesicle transition is observed upon temperature jump. ► The sizes of vesicles are not dictated by those of the pre-annealed discs.

Keywords: Nanodisc; Low temperature annealing; Unilamellar vesicle; Spontaneous formation; Self-assemble; Reaction limited coalescence

Approximate calculation and experimental derivation of native isoelectric points of membrane protein complexes of Arabidopsis chloroplasts and mitochondria by Christof Behrens; Kristina Hartmann; Stephanie Sunderhaus; Hans-Peter Braun; Holger Eubel (pp. 1036-1046).

Electric charges are important intrinsic properties of proteins. They directly affect functionality and also mediate interactions with other molecules such as cofactors, substrates and regulators of enzymatic activity, with lipids as well as other proteins. As such, analysis of the electric properties of proteins gives rise to improved understanding of the mechanism by which proteins fulfil their specific functions. This is not only true for singular proteins but also applies for defined assemblies of proteins, protein complexes and supercomplexes. Charges in proteins often are a consequence of the presence of basic and acidic amino acid residues within polypeptide chains. In liquid phase, charge distributions of proteins change in response to the pH of their environment. The interdependence of protein charge and the surrounding pH is best described by the isoelectric point, which is notoriously difficult to obtain for native protein complexes. Here, experimentally derived native isoelectric points (npIs) for a range mitochondrial and plastid protein complexes are provided. In addition, for four complexes, npIs were calculated by a novel approach which yields results largely matching the experimental npIs.Display Omitted► The native pI of membrane protein complexes is difficult to analyse ► The native pI has functional implications ► Membrane protein complexes for mitochondria and chloroplasts were analysed ► Native pIs were calculated for 2 membrane protein complexes and ATP-synthase F1 subcomplexes with improved accuracy ► Native pIs were also determined experimentally by liquid phase isoelectric focusing

Keywords: Abbreviations; AMPSO; N-(1,1-Dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid; BN; blue-native; CMC; critical micellar concentration; ESI; electro spray ionisation; FFE; free-flow electrophoresis; HEPES; 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid; HPMC; (Hydroxypropyl)methyl cellulose; ICS; intercristal space; IEF; isoelectric focussing; LC; liquid chromatography; LHCII; light-harvesting complex II; MS; mass-spectrometry; nIEF; native isoelectric focussing; npI; native isoelectric point; OPM; Orientation of Proteins in Membranes database; OXPHOS; oxidative phosphorylation; PAGE; polyacrylamide gel electrophoresis; PDB; Protein Data Bank; pI; isoelectric point; SPADNS; 2-(4-Sulfophenylazo)-1,8-dihydroxy- 3,6-naphthalenedisulfonic acid; TOF; time of flightMembrane protein complexes; Ionisable amino acids; Native isoelectric point; Mitochondria; Chloroplasts; Free flow electrophoresis

Electrical potentiation of the membrane permeabilization by new peptides with anticancer properties by Victor V. Lemeshko (pp. 1047-1056).

New polycationic peptides were designed on the basis of 16-mer and 14-mer fragments of the peptide BTM-P1, derived from the Cry11Bb protoxin. The peptides caused mitochondrial, but not red blood cell membrane permeabilization. Conjugation of the cell penetrating hepta-arginine vector to their N- or C-termini through two glycine residues resulted in more active peptides, which also permeabilized the red blood cells with a relatively high plasma membrane potential generated in the presence of valinomycin. The efficiency of the peptides was remarkably higher in the lower ionic strength media. The capability of the plasma membrane permeabilization of the normal red blood cells by the designed conjugated peptides and by known anticancer peptide R7-KLA was also strongly potentiated by the external electrical pulses applied to the cell suspension. These results open the new avenues of the local destruction of solid tumors using the combined “peptide-–electrical pulses” synergistic treatment. The designed peptides were active against the human leukemia Jurkat cells but not against the normal wild type CHO cells.Display Omitted► The hepta-arginine vector was conjugated to natural fragments of the Cry11Bb protoxin. ► Conjugated peptides were highly active in the permeabilization of isolated mitochondria. ► External electrical pulses increased the plasma membrane permeabilization by the peptides. ► The designed peptides selectively killed Jurkat cells, but not CHO cells.

Keywords: Abbreviations; RBCs; red blood cells; DNP; 2,4-dintrophenol; P7-27; IYLATALAKWALKQGF; P7-26; IYLATALAKWALKQ; P7-4; RRRRRRR-GG-IYLATALAKWALKQGF; P7-5; IYLATALAKWALKQGF-GG-RRRRRRR; P7-6; RRRRRRR-GG-IYLATALAKWALKQ; P7-7; IYLATALAKWALKQ-GG-RRRRRRR; R7-KLA; RRRRRRRGGKLAKLAKKLAKLAK; KLA-R7; KLAKLAKKLAKLAK-GG-RRRRRRRAnticancer peptide; Membrane permeabilization; Mitochondrion; Red blood cell; Cell electroporation

Does the lipid environment impact the open-state conductance of an engineered β-barrel protein nanopore? by Noriko Tomita; Mohammad M. Mohammad; David J. Niedzwiecki; Makoto Ohta; Liviu Movileanu (pp. 1057-1065).

Using rational membrane protein design, we were recently able to obtain a β-barrel protein nanopore that was robust under an unusually broad range of experimental circumstances. This protein nanopore was based upon the native scaffold of the bacterial ferric hydroxamate uptake component A (FhuA) of Escherichia coli. In this work, we expanded the examinations of the open-state current of this engineered protein nanopore, also called FhuA ΔC/Δ4L, employing an array of lipid bilayer systems that contained charged and uncharged as well as conical and cylindrical lipids. Remarkably, systematical single-channel analysis of FhuA ΔC/Δ4L indicated that most of its biophysical features, such as the unitary conductance and the stability of the open-state current, were not altered under the conditions tested in this work. However, electrical recordings at high transmembrane potentials revealed that the presence of conical phospholipids within the bilayer catalyzes the first, stepwise current transition of the FhuA ΔC/Δ4L protein nanopore to a lower-conductance open state. This study reinforces the stability of the open-state current of the engineered FhuA ΔC/Δ4L protein nanopore under various experimental conditions, paving the way for further critical developments in biosensing and molecular biomedical diagnosis.Display Omitted► This paper shows the impact of lipid environment on a beta-barrel protein. ► This work demonstrates the unusual stability of the open-state current. ► Conical lipids catalyze the nanopore closure at greater transmembrane potentials.

Keywords: Abbreviations; DPhPC; 1,2-diphytanoyl-; sn; -glycerophosphatidylcholine; DPhPE; 1,2-diphytanoyl-; sn; -glycero-3-phosphoethanolamine; DPhPS; 1,2-diphytanoyl-; sn; -glycero-3-phospho-L-serine; SM; N-palmitoyl-D-erythro-sphingosylphosphorylcholine; ECTPL; E. coli; total polar lipid extract; FhuA; Ferric hydroxamate uptake component A; FhuA ΔC/Δ4L; Deletion mutant of FhuA lacking the plug domain (C) and four extracellular loops (4L) PE, Phosphatidylethanolamine; PG; Phosphatidylglycerol; CL; Cardiolipin; Bcon; Planar lipid bilayer that includes conically-shaped lipids; Bcyl; Planar lipid bilayer that includes only cylindrically-shaped lipids; FCT; First closure time; NCSS; Normalized current step size; NFCT; Normalized first closure time; VDAC; Voltage-dependent anion channel E. coli; FhuA; Planar lipid bilayer; Dimensionless shape factor; Channel closure; Single-channel recording; Lipid effect

Structural and functional studies on a proline-rich peptide isolated from swine saliva endowed with antifungal activity towards Cryptococcus neoformans by Stefania Conti; Giorgia Radicioni; Tecla Ciociola; Renato Longhi; Luciano Polonelli; Rita Gatti; Tiziana Cabras; Irene Messana; Massimo Castagnola; Alberto Vitali (pp. 1066-1074).

A proline-rich peptide of 2733Da, isolated from pig parotid granule preparations was tested against different pathogenic fungi. It showed interesting antifungal activity towards a clinical isolate of Cryptococcus neoformans, with an EC₅₀ of 2.2μM. Neither cytotoxic nor haemolytic effects were observed towards mammalian cells. Circular dichroism and infrared spectroscopic studies showed that the peptide adopted a combination of polyproline type-II, β-turn and unordered conformations at physiological temperatures. Temperature dependent experiments evidenced a tendency to adopt a polyproline-II helix conformation. From experiments with lipid vesicles, Neutral Red Uptake (NRU), haemolytic assays, and confocal microscopy studies, it could be hypothesized that the peptide may exert its antifungal effect by interacting with an intracellular target rather than through membrane damage.Display Omitted► A proline-rich peptide has been isolated from swine saliva possessing antifungal activity. ► It is not cytoxocic towards human control cells. ► It does not show membrane damaging effects, but it is internalized within yeast cells. ► Its structural chracteristcs suggest that this peptide may act through the interaction with an intracellular target.

Keywords: Abbreviations; SPPS; (Solid Phase Peptide Synthesis); MALDI-TOF; (Matrix Assisted Laser Desorption Ionization – Time of flight); CD; (Circular Dichroism); ATR/FT-IR; (Attenuated Total Reflectance/Fourier Transform-Infra Red); PDA; (Polydiacetylene)Proline-rich peptide; Antifungal; Circular dichroism; Membrane integrity; Polyproline-II; Cryptococcus neoformans

Protochlorophyllide and protochlorophyll in model membranes — An influence of hydrophobic side chain moiety by Beata Mysliwa-Kurdziel; Jerzy Kruk; Kazimierz Strzałka (pp. 1075-1082).

In the present work, a comparative study of protochlorophyllide- and protochlorophyll-lipid interaction was performed on liposomes prepared from phospholipids and galactolipids, which had a pigment content varying from 0.1 to 4mol%. The incorporation of pigment molecules into the lipid bilayer and pigment–pigment interactions were investigated. Protochlorophyllide entered the lipid bilayer spontaneously and showed fluorescence spectra characteristic of its monomers. Similar spectra were observed for protochlorophyll where its concentration was low. However, the fluorescence maxima of protochlorophyll monomers were blue-shifted compared to those of protochlorophyllide by about 5nm. Protochlorophyll at high concentrations formed transient aggregates that showed an additional fluorescence band with a maximum at around 685nm, especially in liposomes prepared from phospholipids. For both compounds, the Stern–Volmer constant for KI quenching was much lower in liposomes than in solution, which confirmed the incorporation of these compounds into the lipid bilayer. Two populations of protochlorophyll that differed in their accessibility to quenching by KI were determined, and the proportions between them for different lipids are discussed. Protochlorophyllide showed such heterogeneity only in DPPC membranes. Quenching with 5- and 16-SASL revealed a localization of the porphyrin ring of both Pchl and Pchlide in the polar headgroup area of the lipid bilayer. The side chain of protochlorophyll forced these molecules to localize deeper in the bilayer in the case of DPPC in gel phase.Display Omitted► We studied protochlorophyll and protochlorophyllide in liposomes ► Protochlorophyllide and protochlorophyll enter the bilayer of galactolipids liposomes ► Protochlorophyll in phospholipid vesicles formed transient aggregates ► The porphyrin macrocycle was found in the lipid headgroup area of liposomes ► The side chain forced protochlorophyll molecule to localize deeply in the bilayer

Keywords: Abbreviations; DGDG; digalactosyldiacylglycerol; DPPC; dipalmitoylphosphatidylcholine; LPOR; light-dependent protochlorophyllide oxidoreductase; EYL; egg yolk lecithin; MGDG; monogalactosyldiacylglycerol; n-SASL; spin-labeled stearic acid with a nitroxyl group at the n; ^th; carbon atom of the acyl chain; Pchl; protochlorophyll; Pchlide; protochlorophyllide; PLB; prolamellar body; PT; prothylakoids; SUV; small unilamellar liposomesFluorescence spectrum; Fluorescence lifetime; Fluorescence quenching; Liposome protochlorophyll; Protochlorophyllide

NMR solution structure of C2 domain of MFG-E8 and insights into its molecular recognition with phosphatidylserine by Hong Ye; Baihong Li; Vivekanandan Subramanian; Bo-Hwa Choi; Yu Liang; Amaravadhi Harikishore; Goutam Chakraborty; Kwanghee Baek; Ho Sup Yoon (pp. 1083-1093).

MFG-E8 (also known as lactadherin), which is a secreted glycoprotein from a variety of cell types, possesses two EGF domains and tandem C domains with sequence homology to that of blood coagulation proteins factor V and factor VIII. MFG-E8 binds to phosphatidylserine (PS) in membranes with high affinity. We have recently shown that the C2 domain of MFG-E8 bears more specificity toward PS when compared with phosphatidylcholine (PC), another phospholipid thought to be involved in the immune function of phagocytes. In our current study, we have determined the solution structure of the C2 domain by nuclear magnetic resonance (NMR) spectroscopy, and characterized the molecular basis of binding between the C2 domain and PS by³¹P-NMR spectroscopy. Furthermore, we also verified that that positively charged and aromatic residues clustered in loops 1–3 of the C2 domain play key roles in recognizing PS in apoptotic cells.Display Omitted► NMR solution structure of MFG-E8 C2 domain ► Electrostatic potential map of the C2 shows the clustered positive charges in the PS-binding site. ► K45, R79, and R146 residues of C2 are involved in the PS interaction. ► Mutation of the positively charged residues results in a dramatic decrease in the C2's PS binding.

Keywords: MFG-E8; C2 domain; Phosphatidylserine; NMR; Phagocytosis; Apoptosis

Sensing hydration and behavior of pyrene in POPC and POPC/cholesterol bilayers: A molecular dynamics study by Luís M.S. Loura; António M.T. Martins do Canto; Jorge Martins (pp. 1094-1101).

Molecular dynamics (MD) simulations of bilayers of 1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC) with varying amounts of cholesterol (0, 5, 20, and 40mol%) were carried out in the absence and presence of inserted pyrene molecules. Both fluorophore and bilayer parameters were computed, for characterization of probe location and dynamics, as well as its effects on the host bilayer. In agreement with previous studies in fluid disordered bilayers, pyrene prefers to be located in the hydrophobic acyl chain region of POPC bilayers, close to the glycerol group of lipid molecules and causes ordering of the lipid acyl chains. However, incorporation of pyrene in binary POPC/cholesterol bilayers decreases the acyl chain order parameter (especially near the end of the chains), opposing the ordering effect of cholesterol. These effects are modest and mainly felt locally. Significantly, as the bilayer is enriched with cholesterol, the relative position of pyrene and the POPC carbonyl and phosphocholine groups is invariant, and the local water density around the probe decreases. This work clarifies and supports the cautious use of pyrene Ham effect to effectively measure equivalent polarity in lipid bilayers. Within the time scale of the MD simulations, which is of the magnitude of the fluorescence lifetime of pyrene, the thermally averaged polarity of lipid bilayers is nearly out of influence of spurious uncertainty in the transverse location of pyrene in the bilayers. This renders the values of equivalent polarity measurements through the pyrene Ham effect more reliable and reproducible than previously expected.Display Omitted► Pyrene was simulated in POPC bilayers with variable amounts of cholesterol (Chol). ► Effects of pyrene on the bilayer structure and dynamics are globally small. ► Pyrene locates in the upper acyl chain region, and orders acyl chains in pure POPC. ► However, pyrene locally disorders Chol-rich bilayers, opposing the effect of Chol. ► The bilayer polarity sensed by the pyrene Ham effect is more precise than expected.

Keywords: Abbreviations; DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phosphocholine; MD; molecular dynamics; POPC; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; RDF; radial distribution functionFluorescent membrane probe; Ham effect; Lipid bilayer; Molecular dynamics simulation; Pyrene; Py; polarity scale

Actin filaments attachment at the plasma membrane in live cells cause the formation of ordered lipid domains by Jelena Dinic; Parham Ashrafzadeh; Ingela Parmryd (pp. 1102-1111).

The relationship between ordered plasma membrane nanodomains, known as lipid rafts, and actin filaments is the focus of this study. Plasma membrane order was followed in live cells at 37°C using laurdan and di-4-ANEPPDHQ to report on lipid packing. Disrupting actin polymerisation decreased the fraction of ordered domains, which strongly argue that unstimulated cells have a basal level of ordered domains. Stabilising actin filaments had the opposite effect and increased the proportion of ordered domains. Decreasing the plasma membrane level of 4-phosphate-inositides lowers the number of attachment points for actin filaments and reduced the proportion of ordered domains. Aggregation of plasma membrane molecules, both lipid raft and non-lipid raft markers, lead to the formation of ordered domains. The increase in ordered domains was correlated with an increase in actin filaments just beneath the plasma membrane. In live cell plasma membrane blebs, which are detached from the underlying actin filaments, the fraction of ordered domains was low and GM1 could not be patched to form ordered domains. We conclude that ordered domains form when actin filaments attach to the plasma membrane. This downplays lipid-lipid interactions as the main driving force behind the formation of ordered membrane domains in vivo, giving greater prominence to membrane-intracellular filament interactions.Display Omitted► Ordered plasma membrane domains exist in resting cells. ► Ordered membrane domains form when actin filaments attach to the plasma membrane. ► Phosphoinositides link actin dynamics to plasma membrane order. ► Intracellular actin dynamics are reflected in the outer leaflet of the plasma membrane. ► Molecular patching induces actin polymerisation and ordered domain formation.

Keywords: Abbreviations; CT-B; cholera toxin subunit B; DOPC; dioleoyl phosphatidyl choline; DRMs; detergent-resistant membranes; GP; generalised polarisation; Jas; jasplakinolide; Lat B; latrunculin B; laurdan; 6-dodecanoyl-2-dimethyl-aminonaphthalene; ld; liquid-disordered; lo; liquid-ordered; LUVs; large unilamellar vesicles; PAO; phenylarsine oxide; PI(4,5)P2; phosphatidylinositol 4,5-bisphosphateActin; di-4-ANEPPDHQ; Laurdan; Lipid raft; Liquid ordered phase; Phosphoinositide

Molecular simulations suggest how a branched antimicrobial peptide perturbs a bacterial membrane and enhances permeability by Jianguo Li; Shouping Liu; Rajamani Lakshminarayanan; Yang Bai; Konstantin Pervushin; Chandra Verma; Roger W. Beuerman (pp. 1112-1121).

A covalently, branched antimicrobial peptide (BAMP) B2088 demonstrating enhanced antimicrobial effects and without additional toxicity when compared to its linear counterpart, has been developed. Atomistic molecular dynamics simulations have been used to investigate the mode of interaction of B2088 with model bacterial and mammalian membranes. These simulations suggest that both long-range electrostatic interactions and short-range hydrogen bonding play important roles in steering B2088 toward the negatively charged membranes. The reason why B2088 is selective towards the bacterial membrane is postulated to be the greater density of negative charges on the bacterial membrane which enables rapid accumulation of B2088 on the bacterial membrane to a high surface concentration, stabilizing it through excess hydrogen bond formation. The majority of hydrogen bonds are seen between the side chains of the basic residues (Arg or Lys) with the PO4 groups of lipids. In particular, formation of the bidentate hydrogen bonds between the guanidinium group of Arg and PO4 groups are found to be more favorable, both geometrically and energetically. Moreover, the planar gaunidinium group and its hydrophobic character enable the Arg side chains to solvate into the hydrophobic membrane. Structural perturbation of the bacterial membrane is found to be concentration dependent and is significant at higher concentrations of B2088, resulting in a large number of water translocations across the bacterial membrane. These simulations enhance our understanding of the action mechanism of a covalently branched antimicrobial peptide with model membranes and provide practical guidance for the design of new antimicrobial peptides.Display Omitted► The mode of interaction of a branched AMP B2088 with model membranes was studied. ► The simulation predictions are consistent with the surface activity model. ► High concentration of B2088 on bacterial membrane leads to enhanced permeability. ► The bidentate H bonds between Arg and PO4 groups of lipids favor binding.

Keywords: Branched antimicrobial peptides; Model bacterial membrane; Molecular dynamics simulations

Effect of glucosylceramide on the biophysical properties of fluid membranes by Ana R.P. Varela; Goncalves da Silva Amélia M.P.S. Gonçalves da Silva; Alexander Fedorov; Anthony H. Futerman; Manuel Prieto; Liana C. Silva (pp. 1122-1130).

Glucosylceramide (GlcCer), a relevant intermediate in the pathways of glycosphingolipid metabolism, plays key roles in the regulation of cell physiology. The molecular mechanisms by which GlcCer regulates cellular processes are unknown, but might involve changes in membrane biophysical properties and formation of lipid domains. In the present study, fluorescence spectroscopy, confocal microscopy and surface pressure–area (π–A) measurements were used to characterize the effect of GlcCer on the biophysical properties of model membranes. We show that C16:0-GlcCer has a high tendency to segregate into highly ordered gel domains and to increase the order of the fluid phase. Monolayer studies support the aggregation propensity of C16:0-GlcCer. π–A isotherms of single C16:0-GlcCer indicate that bilayer domains, or crystal-like structures, coexist within monolayer domains at the air–water interface. Mixtures with POPC exhibit partial miscibility with expansion of the mean molecular areas relative to the additive behavior of the components. Moreover, C16:0-GlcCer promotes morphological alterations in lipid vesicles leading to formation of flexible tubule-like structures that protrude from the fluid region of the bilayer. These results support the hypothesis that alterations in membrane biophysical properties induced by GlcCer might be involved in its mechanism of action.Display Omitted► GlcCer segregates into highly ordered gel domains ► GlcCer increases the order of the fluid phase ► GlcCer promotes morphological alterations on the membrane ► GlcCer biological actions may be driven by changes in membrane properties

Keywords: Abbreviations; C16:0-Cer; N-palmitoyl-; d; -; erythro; -sphingosine; C24:0-Cer; N; -lignoceroyl-; d; -; erythro; -sphingosine; C24:1-Cer; N; -nervonoyl-; d; -; erythro; -sphingosine; C16:0-GlcCer; d; -glucosyl-ß-1,1′ N-palmitoyl-; d; -; erythro; -sphingosine; Cer; Ceramide; Chol; Cholesterol; DPH; 1,6-diphenyl-1,3,5-hexatriene; FAPP2; 4-phosphate adaptor protein-2; GalCer; Galactosylceramide; GCS; GlucosylceramideSynthase; GlcCer; Glucosylceramide; GSL; Glycosphingolipid; GUV; Giant unilamellar vesicles; MMA; Mean Molecular Area; MLV; Multilamellar Vesicles; NBD-DPPE; 1,2-dipalmitoyl-; sn; -glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl); POPC; 1-palmitoyl-2-oleoyl-; sn; -glycerol-3-phosphocholine; Rho-DOPE; 2-Dioleoyl-; sn; -Glycero-3-Phosphoethanolamine-N-(LissamineRhodamine B Sulfonyl); SL; Sphingolipids; SOPC; 1-stearoyl-2-oleoyl-; sn; -glycero-3-phosphocholine; T; _m; Main Transition Temperature; t; -PnA; trans; -parinaric acid (octadeca-9,11,13,15-tetraenoic acid)Glucosylceramide; Lipid gel domains; Lipid tubules; Membrane order

Identification of blocker binding site in mouse TRESK by molecular modeling and mutational studies by Songmi Kim; Yuno Lee; Hyun-Min Tak; Hye-Jin Park; Young-sik Sohn; Swan Hwang; Jaehee Han; Dawon Kang; Keun Woo Lee (pp. 1131-1142).

TWIK (tandem-pore domain weak inward rectifying K⁺)-related spinal cord K⁺ channel, TRESK, a member of the tandem-pore domain K⁺ channel family, is the most recently cloned K_2P channel. TRESK is highly expressed in dorsal root ganglion neuron, a pain sensing neuron, which is a target for analgesics. In this study, a reliable 3D structure for transmembrane (TM) region of mouse TRESK (mTRESK) was constructed, and then the reasonable blocker binding mode of the protein was investigated. The 3D structure of the mTRESK built by homology modeling method was validated with recommend value of stereochemical quality. Based on the validated structure, K⁺ channel blocker-bound conformation was obtained by molecular docking and 5ns MD simulation with DPPC lipid bilayer. Our docking study provides the plausible binding mode of known blockers with key interacting residues, especially, F156 and F364. Finally, these modeling results were verified by experimental study with mutation from phenylalanine to alanine (F156A, F364A and F156A/F364A) at the TM2 and TM4. This is the first modeling study for TRESK that can provide structural information of the protein including ligand binding information. These results can be useful in structure based drug design for finding new blockers of the TRESK as potential therapeutic target of pain treatment.Display Omitted► The reliable 3D structure for transmembrane region of mTRESK was successfully constructed. ► Reasonable blocker binding mode of the blockers with the protein was investigated. ► Key interacting residues were verified by experimental mutation study. ► Our study can provide very important structural knowledge of the protein including ligand binding information.

Keywords: TRESK (TWIK-related spinal cord K; ⁺; channel); K; _2P; channel; Homology modeling; Blocker binding site; Molecular docking simulation; Molecular dynamics simulation

Modeling the surface of campylobacter fetus: Protein surface layer stability and resistance to cationic antimicrobial peptides by James M.D. Roberts; Lori L. Graham; Bonnie Quinn; David A. Pink (pp. 1143-1152).

Campylobacter fetus is a Gram negative bacterium recognized for its virulence in animals and humans. This bacterium possesses a paracrystalline array of high molecular weight proteins known as surface-layer proteins covering its cell surface. A mathematical model has been made of the outer membrane of this bacterium, both with its surface-layer proteins (S+) and without (S−). Monte Carlo computer simulation was used to understand the stability of the surface-layer protein structure as a function of ionic concentration. The interactions of an electrically-charged antimicrobial agent, the cationic antimicrobial peptide protamine, with surface-layer proteins and with the lipopolysaccharides of the outer membrane were modeled and analyzed. We found that (1) divalent ions stabilize the surface-layer protein array by reducing the fluctuations perpendicular and parallel to the membrane plane thereby promoting adhesion to the LPS region. This was achieved via (2) divalent ions bridging the negatively-charged LPS Core. The effect of this bridging is to bring individual Core regions closer together so that the O-antigens can (3) increase their attractive van der Waals interactions and “collapse” to form a surface with reduced perpendicular fluctuations. These findings provide support for the proposal of Yang et al. [1]. (4) No evidence for a significant increase in Ca2⁺ concentration in the region of the surface-layer protein subunits was observed in S+ simulations compared to S− simulations. (5) We predicted the trends of protamine MIC tests performed on C. fetus and these were in good agreement with our experimental results.Top to bottom, left to right: Distribution of LPS moieties perpendicular to the membrane ( z-axis). Distribution of protein moieties in the membrane plane. Ion distribution along the z-axis. Protamine distribution along the z-axis.Display Omitted► A coarse-grained model of the outer membrane of Campylobacter fetus ► Monte Carlo simulation to understand stability of the surface-layer protein structure ► Predicted trends of protamine MIC tests in agreement with experimental results

Keywords: Campylobacter fetus outer membrane; Stability of surface layer protein array; Coarsegrained modeling and Monte Carlo computer simulation; Effect of mono- and di-valent ion; MIC measurement

The antifungal activity and membrane-disruptive action of dioscin extracted from Dioscorea nipponica by Jaeyong Cho; Hyemin Choi; Juneyoung Lee; Mi-Sun Kim; Ho-Yong Sohn; Dong Gun Lee (pp. 1153-1158).

Dioscin is a kind of steroidal saponin isolated from the root bark of wild yam Dioscorea nipponica. We investigated the antifungal effect of dioscin against different fungal strains and its antifungal mechanism(s) in Candida albicans cells. Using the propidium iodide assay and calcein-leakage measurement, we confirmed that dioscin caused fungal membrane damage. Furthermore, we evaluated the ability of dioscin to disrupt the plasma membrane potential, using 3,3′-dipropylthiadicarbocyanine iodide [DiSC₃(5)] and bis-(1,3-dibarbituric acid)-trimethine oxanol [DiBAC₄(3)]. Cells stained with the dyes had a significant increase in fluorescent intensity after exposure to dioscin, indicating that dioscin has an effect on the membrane potential. To visualize the effect of dioscin on the cell membrane, we synthesized rhodamine-labeled giant unilamellar vesicles (GUVs) mimicking the outer leaflet of the plasma membrane of C. albicans. As seen in the result, the membrane disruptive action of dioscin caused morphological change and rhodamine leakage of the GUVs. In three-dimensional contour-plot analysis using flow cytometry, we observed a decrease in cell size, which is in agreement with our result from the GUV assay. These results suggest that dioscin exerts a considerable antifungal activity by disrupting the structure in membrane after invading into the fungal membrane, resulting in fungal cell death.Display Omitted► The antifungal effect of dioscin was investigated. ► The effect of dioscin on Candida albicans membrane was identified. ► Dioscin exerted antifungal effect via membrane disruption, resulting in cell death.

Keywords: Abbreviations; Dioscin; [3-O-α-; l; -rhamnopyranosyl-(1,4)-[α-; l; -rhamnopyranosyl-(1,2)]-β-; d; -glucopyranosyl-diosgenin]; MTT; 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2; H; -tetrazolium bromide; LUVs; large unilamellar vesicles; PC; phosphatidylcholine; PE; phosphatidylethanolamine; PI; phosphatidylinositol; DiSC; ₃; (5); 3,3′-dipropylthiacarbocyanine; DiBAC; ₄; (3); bis-(1,3-dibutylbarbituric acid) trimethine oxonol; GUV; giant unilamellar vesicle; ITO; indium tin oxideDioscin; Dioscorea nipponica; Antifungal activity; Mechanism

P-glycoprotein is fully active after multiple tryptophan substitutions by Douglas J. Swartz; Joachim Weber; Ina L. Urbatsch (pp. 1159-1168).

P-glycoprotein (Pgp) is an important contributor to multidrug resistance of cancer. Pgp contains eleven native tryptophans (Trps) that are highly conserved among orthologs. We replaced each Trp by a conservative substitution to determine which Trps are important for function. Individual Trp mutants W44R, W208Y, W132Y, W704Y and W851Y, situated at the membrane surface, revealed significantly reduced Pgp induced drug resistance against one or more fungicides and/or reduced mating efficiencies in Saccharomyces cerevisiae. W158F and W799F, located in the intracellular coupling helices, abolished mating but retained resistance against most drugs. In contrast, W228F and W311Y, located within the membrane, W694L, at the cytoplasmic membrane interface, and W1104Y in NBD2 retained high levels of drug resistance and mating efficiencies similar to wild-type Pgp. Those were combined into pair (W228F/W311Y and W694L/W1104Y) and quadruple (W228F/W311Y/W694L/W1104Y) mutants that were fully active in yeast, and could be purified to homogeneity. Purified pair and quad mutants exhibited drug-stimulated ATPase activity with binding affinities very similar to wild-type Pgp. The combined mutations reduced Trp fluorescence by 35%, but drug induced fluorescence quenching was unchanged from wild-type Pgp suggesting that several membrane-bound Trps are sensitive to drug binding. Overall, we conclude that Trps at the membrane surface are critical for maintaining the integrity of the drug binding sites, while Trps in the coupling helices are important for proper interdomain communication. We also demonstrate that functional single Trp mutants can be combined to form a fully active Pgp that maintains drug polyspecificity, while significantly reducing intrinsic fluorescence.Display Omitted► Substitution of the eleven native Trps in the P-glycoprotein multidrug transporter. ► Several Trps at the membrane interface are critical for substrate polyspecificity. ► Trps in the coupling helices are important for interdomain communication. ► Single Trp mutants can be combined while maintaining drug polyspecificity. ► Multi-Trp mutant protein is fully active with reduced intrinsic fluorescence.

Keywords: Abbreviations; Pgp; P-glycoprotein; ABC; ATP binding cassette; TMD; Transmembrane domain; NBD; Nucleotide binding domain; Trp; Tryptophan; DDM; n-Dodecyl-β-; d; -maltopyranoside; NATA; N; -acetyl-; l; -tryptophanamide; PMPC; 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholineP-glycoprotein; Native tryptophans; Membrane bilayer; Conservative tryptophan substitutions; Drug binding sites; Polyspecificity

Linoleic acid induces opening of connexin26 hemichannels through a PI3K/Akt/Ca2⁺-dependent pathway by Vania Figueroa; Saez Pablo J. Sáez; José D. Salas; Daniela Salas; Oscar Jara; Martinez Agustín D. Martínez; Saez Juan C. Sáez; Mauricio A. Retamal (pp. 1169-1179).

Connexin hemichannel (Cx HC) opening is involved in physiological and pathological processes, allowing the cellular release of autocrine/paracrine signaling molecules. Linoleic acid (LA) is known to modulate the functional state of connexin46 (Cx46) HCs. However, the molecular mechanism involved in this effect, or whether LA affects HCs constituted of other connexins, remains unknown. Here, we report the effects of LA on HCs in HeLa cells that express Cx26, one of the main Cxs in the cochlear sensory epithelium. Cx26 HC activity (dye uptake) was increased in a concentration-dependent manner by bath application of LA and inhibited by HC blockers. Moreover, intracellular BAPTA, a Ca2⁺ chelator, and PI3K/AKT inhibitors were found to reduce the LA-induced Cx26 HC opening, suggesting that the LA effect is mediated by an increase of free intracellular Ca2⁺ concentration and activation of the PI3K/Akt-dependent pathway. The LA-induced increase in free intracellular Ca2⁺ concentration was mainly due to Ca2⁺ influx through Cx26 HCs. In addition, the involvement ofSH groups was ruled out, because dithiothreitol (DTT) did not block the LA-induced dye uptake. LA also increased the membrane current mediated by Cx26 HCs expressed in Xenopus oocytes and the dye uptake in HeLa cells expressing Cxs 32, 43 or 45. Since LA is an essential polyunsaturated fatty acid, its effect on HCs might be relevant to cell growth as well as to cellular functions of differentiated cells such as audition.Display Omitted► LA increases the activity of HCs formed by Cxs 26, 32, 43 and 45. ► LA induces a rise of intracellular free Ca2⁺ and activation of a PI3K/Akt-dependent pathway. ► Cx26 HCs mediate the Ca2⁺ influx to the cells in response to LA. ► Cx26 HCs are sensitive to a reducing environment.

Keywords: Abbreviations; Cxs; connexins; GJCs; gap junction channels; HCs; hemichannels; LA; linoleic acid; Etd; ethidium; DTT; dithiothreitolPolyunsaturated fatty acid; Hemichannel; Cx26; Intracellular calcium; Hearing loss

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BBA - Biomembranes (v.1828, #3)