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BBA - Biomembranes (v.1808, #9)
The interaction of cannabinoid receptor agonists, CP55940 and WIN55212-2 with membranes using solid state2H NMR by Xiaoyu Tian; Spiro Pavlopoulos; De-Ping Yang; Alexandros Makriyannis (pp. 2095-2101).
Two key commonly used cannabinergic agonists, CP55940 and WIN55212-2, are investigated for their effects on the lipid membrane bilayer using2H solid state NMR, and the results are compared with our earlier work with delta-9-tetrahydrocannabinol (Δ9-THC). To study the effects of these ligands we used hydrated bilayers of dipalmitoylphosphatidylcholine (DPPC) deuterated at the 2′ and 16′ positions of both acyl chains with deuterium atoms serving as probes for the dynamic and phase changes at the membrane interface and at the bilayer center respectively. All three cannabinergic ligands lower the phospholipid membrane phase transition temperature, increase the lipid sn-2 chain order parameter at the membrane interface and decrease the order at the center of the bilayer.Our studies show that the cannabinoid ligands induce lateral phase separation in the lipid membrane at physiological temperatures. During the lipid membrane phase transition, the cooperative dynamic process whereby the C-2H segments at the interface and center of the bilayer spontaneously reach the fast exchange regime (2H NMR timescale) is distinctively modulated by the two cannabinoids. Specifically, CP55940 is slightly more efficient at inducing liquid crystalline-type2H NMR spectral features at the membrane interface compared to WIN55212-2. In contrast, WIN55212-2 has a far superior ability to induce liquid crystalline-type spectral features at the center of the bilayer, and it increases the order parameter of the sn-1 chain in addition to the sn-2 chain of the lipids. These observations suggest the cannabinoid ligands may influence lipid membrane domain formations and there may be contributions to their cannabinergic activities through lipid membrane microdomain related mechanisms. Our work demonstrates that experimental design strategies utilizing specifically deuterium labeled lipids yield more detailed insights concerning the properties of lipid bilayers.► Lipid membrane effects of two cannabinoid agonists, WIN55212-2 and CP55940, are investigated. ► DPPC lipid membrane with specific deuterium labels provides extra details for solid state2H NMR studies. ► The cannabinoid agonists induce lipid lateral phase separation and promote microdomain formation. ► The cooperative process of the lipid acyl chain C-2H segment may be modulated by the cannabinoids. ► The agonists lower the lipid phase transition temperature and modulate the chain order distinctively.
Keywords: Cannabinoid receptor agonist; Win55212-2; CP55940; THC; Lipid membrane phase transition; Drug membrane interaction; Lipid domain; Solid state NMR
Structure of the lipodepsipeptide syringomycin E in phospholipids and sodium dodecylsulphate micelle studied by circular dichroism, NMR spectroscopy and molecular dynamics by Massimiliano Anselmi; Tommaso Eliseo; Laura Zanetti-Polzi; Maria Rosaria Fullone; Vincenzo Fogliano; Alfredo Di Nola; Maurizio Paci; Ingeborg Grgurina (pp. 2102-2110).
Syringomycin E (SRE) is a member of a family of lipodepsipeptides that characterize the secondary metabolism of the plant-associated bacteria Pseudomonas syringae pv. syringae. It displays phytotoxic, antifungal and haemolytic activities, due to the membrane interaction and ion channel formation. To gain an insight into the conformation of SRE in the membrane environment, we studied the conformation of SRE bound to SDS micelle, a suitable model for the membrane-bound SRE. In fact, highly similar circular dichroism (CD) spectra were obtained for SRE bound to sodium dodecylsulphate (SDS) and to a phospholipid bilayer, indicating the conformational equivalence of SRE in these two media, at difference with the CD spectrum of SRE in water solution. The structure of SDS-bound SRE was determined by NMR spectroscopy combined with molecular dynamics calculations in octane environment. The results of this study highlight the influence of the interaction with lipids in determining the three-dimensional structure of SRE and provide the basis for further investigations on structural determinants of syringomycin E-membrane interaction.► SDS micelle and phospholipids induce modifications in the three-dimensional structure of syringomycin E. ► Conformation of syringomycin E in SDS micelle was studied by NMR spectroscopy and molecular dynamics calculations.
Keywords: Abbreviations; NMR; Nuclear Magnetic Resonance; MD; Molecular Dynamics; HMQC; Heteronuclear Multiple Quantum Correlation; COSY; Correlation Spectroscopy; TOCSY; Total Correlation Spectroscopy; CD; Circular Dichroism; GROMOS 96; Groningen MoleculaR Simulation; GROMACS; Groningen Machine for Chemical SimulationsSyringomycin E conformation; SDS micelle; Phospholipid; NMR spectroscopy; Molecular dynamics; Antifungal lipodepsipeptide
Binding of subunit E into the A–B interface of the A1AO ATP synthase by Cornelia Hunke; Martin Antosch; Muller Volker Müller; Gruber Gerhard Grüber (pp. 2111-2118).
Two of the distinct diversities of the engines A1AO ATP synthase and F1FO ATP synthase are the existence of two peripheral stalks and the 24kDa stalk subunit E inside the A1AO ATP synthase. Crystallographic structures of subunit E have been determined recently, but the epitope(s) and the strength to which this subunit does bind in the enzyme complex are still a puzzle. Using the recombinant A3B3D complex and the major subunits A and B of the methanogenic A1AO ATP synthase in combination with fluorescence correlation spectroscopy (FCS) we demonstrate, that the stalk subunit E does bind to the catalytic headpiece formed by the A3B3 hexamer with an affinity ( K d) of 6.1±0.2μM. FCS experiments with single A and B, respectively, demonstrated unequivocally that subunit E binds stronger to subunit B ( K d=18.9±3.7μM) than to the catalytic A subunit ( K d=53.1±4.4). Based on the crystallographic structures of the three subunits A, B and E available, the arrangement of the peripheral stalk subunit E in the A–B interface has been modeled, shining light into the A–B–E assembly of this enzyme.Display Omitted► Experimental evidence demonstrates that the stalk subunit E binds to the catalytic A3B3 hexamer. ► Fluorescence correlation spectroscopy reveals that subunit E binds stronger to B than to subunit A. ► The arrangement of the peripheral stalk subunit E in the A–B interface has been modeled.
Keywords: Archaeal ATP synthase; A; 1; A; O; ATP synthase; Methanosarcina mazei; Gö1; Peripheral stalk; Fluorescence correlation spectroscopy; F; 1; F; O; ATP synthase
Amphipathic-Lipid-Packing-Sensor interactions with lipids assessed by atomistic molecular dynamics by Gonzalez-Rubio Paula González-Rubio; Romain Gautier; Catherine Etchebest; Patrick F.J. Fuchs (pp. 2119-2127).
The Amphipathic-Lipid-Packing-Sensor (ALPS) motif targets the protein ArfGAP1 to curved membranes during vesicle formation in the Golgi apparatus. ALPS specifically recognizes lipid packing defects due to the positive curvature of budding vesicles. In this work we assessed the microscopic interactions between ALPS and two phospholipid membranes at different degrees of lipid packing by explicit molecular dynamics (MD). Simulations were performed within loosely packed membranes composed of a mixture of dioleoylphosphatidylcholine (DOPC)/dioleoylglycerol (DOG) at a molar ratio 85:15. Some other simulations were performed in pure DOPC for which lipid packing is tighter. We show that the presence of DOG causes packing defects at the phosphate level and thereby modifies some properties of the bilayer. This leads to a higher hydration of the lipid headgroups. When embedded in a membrane with such defects, ALPS displays a higher degree of conformational flexibility than in a more packed membrane. We propose that lipid packing sensing by ALPS may have an entropic origin and that its flexibility is a key feature.Display Omitted► The Amphipathic-Lipid-Packing-Sensor (ALPS) motif is studied by molecular dynamics. ► Trajectories are performed in pure DOPC and in a mixture DOPC/DOG (85:15 molar ratio). ► The introduction of DOG in a DOPC bilayer induces packing defects. ► ALPS displays more flexibility in a loosely packed membrane. ► ALPS lipid packing sensing capability may have an entropic origin.
Keywords: ALPS motif; Molecular dynamics; Amphiphatic α-helix; DOPC/DOG mixture; Packing defects; Peptide flexibility
Multivalent protein binding in carbohydrate-functionalized monolayers through protein-directed rearrangement and reorientation of glycolipids at the air–water interface by Haifu Zheng; Xuezhong Du (pp. 2128-2135).
Multivalent protein binding plays an important role not only in biological recognition but also in biosensor preparation. Infrared reflection absorption spectroscopy and surface plasmon resonance techniques have been used to investigate concanavalin A (Con A) binding to binary monolayers composed of 1,2-di- O-hexadecyl- sn-glycerol and derived glycolipids with the mannose moieties. The glycolipids in the binary monolayers at the air–water interface underwent both lateral rearrangement and molecular reorientation directed by Con A in the subphase favorable to access of the carbohydrate ligands to protein binding pockets for the formation of multivalent binding sites and the minimization of steric crowding of neighboring ligands for enhanced binding. The amounts of specifically bound proteins in the binary monolayers at the air–water interface were accordingly increased in comparison with those in the initially immobilized monolayers at the air–water interface. The directed rearranged binary monolayers with multivalent protein binding were preserved for the preparation of biosensors.Display Omitted► Con A binding to glycolipid-containing binary monolayers at the air–water interface. ► Protein-directed spatial rearrangement and reorientation of glycolipids. ► Formation of multivalent protein binding and enhancement of binding affinity. ► Immobilization of rearranged binary monolayers for subsequent biosensing.
Keywords: Infrared spectroscopy; Surface plasmon resonance; Aqueous monolayer; Lipid rearrangement; Multivalent protein binding
The effect of aliphatic alcohols on fluid bilayers in unilamellar DOPC vesicles — A small-angle neutron scattering and molecular dynamics study by Klacsova M. Klacsová; M. Bulacu; Kucerka N. Kučerka; Uhrikova D. Uhríková; J. Teixeira; S.J. Marrink; Balgavy P. Balgavý (pp. 2136-2146).
Small-angle neutron scattering and coarse-grained molecular dynamics simulations have been used to determine the structural parameters (bilayer thickness D, polar region thickness D H, interfacial lateral area of the unit cell A UC and alcohol partial interfacial area A CnOH) of fluid dioleoylphosphatidylcholine:dioleoylphosphatidylserine (PCPS, DOPC:DOPS=24.7mol:mol) bilayers in extruded unilamellar vesicles with incorporated aliphatic alcohols (CnOH, n=8–18 is the even number of carbons in alkyl chain). External2H2O/H2O contrast variation experiments revealed that D H decreases as a function of alkyl chain length and CnOH:PCPS molar ratio. Using measurements at single 100%2H2O contrast we found that (i) D decreases with CnOH:PCPS molar ratio and increases with CnOH chain length (at 0.4 molar ratio); (ii) A UC significantly increases already in the presence of shortest CnOH studied (at 0.4 molar ratio), further increase is observed with longer CnOHs and at higher molar ratios; (iii) A CnOH of alcohol molecules in PCPS bilayer increases linearly with the alkyl chain length, A CnOH obtained for CnOHs with n≤10 corresponds to A CnOH≤20Å2 — a value specific for the crystalline or solid rotator phase of alkanes. All these structural modifications induced by studied CnOHs were reproduced in MD simulations. The computational results give an accurate description of the alcohol effects at the molecular level, explaining the experimental data. The anomaly in A CnOH is discussed via the “umbrella” effect described for cholesterol.► Neutron scattering and molecular dynamics study on structural parameters of DOPC+DOPS+CnOH (n=8–18) bilayers. ► Bilayer thickness decreases with CnOH:PCPS molar ratio and increases with CnOH chain length. ► Lateral area of the unit cell significantly increases already in the presence of C8OH. ► Alcohol partial interfacial area in bilayers increases linearly with the alkyl chain length. ► Alcohol partial interfacial area obtained for CnOHs with n≤10 corresponds to A CnOH≤20Å2.
Keywords: Abbreviations; PC; phosphatidylcholine; DOPC; 1,2-dioleoyl-; sn; -glycero-3-phosphatidylcholine; DOPS; 1,2-dioleoyl-; sn; -glycero-3-phosphatidylserine; DMPC; 1,2-dimyristoyl-; sn; -glycero-3-phosphatidylcholine; DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phosphatidylcholine; PCPS; homogeneous mixture of DOPC (96; wt.%) and DOPS (4; wt.%); CnOH; alkan-1-ol (n is the number of carbons in the aliphatic chain); EYPC; egg yolk phosphatidylcholine; SANS; small-angle neutron scattering; SAND; small-angle neutron diffraction; SAXS; small-angle X-ray scattering; SAXD; small-angle X-ray diffraction; WAXD; wide-angle X-ray diffraction; GIXD; grazing incidence X-ray diffraction; MD; molecular dynamics; ULV; unilamellar vesicleAlcohol; Anesthetic; Lipid bilayer; Dioleoylphosphatidylcholine; Small-angle neutron scattering; Coarse-grained simulation
Topography of tyrosine residues and their involvement in peroxidation of polyunsaturated cardiolipin in cytochrome c/cardiolipin peroxidase complexes by Alexandr A. Kapralov; Naveena Yanamala; Yulia Y. Tyurina; Laura Castro; Alejandro Samhan-Arias; Yuri A. Vladimirov; Akihiro Maeda; Andrew A. Weitz; Jim Peterson; Danila Mylnikov; Verónica Demicheli; Verónica Tortora; Judith Klein-Seetharaman; Rafael Radi; Valerian E. Kagan (pp. 2147-2155).
Formation of cytochrome c (cyt c)/cardiolipin (CL) peroxidase complex selective toward peroxidation of polyunsaturated CLs is a pre-requisite for mitochondrial membrane permeabilization. Tyrosine residues – via the generation of tyrosyl radicals (Tyr) – are likely reactive intermediates of the peroxidase cycle leading to CL peroxidation. We used mutants of horse heart cyt c in which each of the four Tyr residues was substituted for Phe and assessed their contribution to the peroxidase catalysis. Tyr67Phe mutation was associated with a partial loss of the oxygenase function of the cyt c/CL complex and the lowest concentration of H2O2-induced Tyr radicals inelectron paramagnetic resonance (EPR) spectra. Our MS experiments directly demonstrated decreased production of CL-hydroperoxides (CL-OOH) by Tyr67Phe mutant. Similarly, oxidation of a phenolic substrate, Amplex Red, was affected to a greater extent in Tyr67Phe than in three other mutants. Tyr67Phe mutant exerted high resistance to H2O2-induced oligomerization. Measurements of Tyr fluorescence, hetero-nuclear magnetic resonance(NMR) and computer simulations position Tyr67 in close proximity to the porphyrin ring heme iron and one of the two axial heme-iron ligand residues, Met80. Thus, the highly conserved Tyr67 is a likely electron-donor (radical acceptor) in the oxygenase half-reaction of the cyt c/CL peroxidase complex.► Cyt c forms complexes with cardiolipin (CL) and catalyzes CL peroxidation. ► Tyrosyl radicals are reactive intermediates of cyt c/CL peroxidase. ► Tyr67 is closest to the heme-iron of cyt c. ► Tyr67 mutant displayed weakest EPR radical signals and lowest CL peroxidation. ► Tyr67 is a likely electron-donor in the oxygenase half-reaction of cyt c/CL complex.
Keywords: Abbreviations; CL; cardiolipin; TOCL; 1,1′,2,2′-tetraoleoylcardiolipin; TLCL; 1,1′,2,2′-tetralinoleoylcardiolipin; DOPC; 1,2-dioleoyl-; sn; -glycero-3-phosphocholine; cyt c; cytochrome c; wt; wild type; Tyr; tyrosyl radicals; GndCl; guanidine hydrochloride; DTPA; diethylenetriaminepentaacetic acid; H; 2; O; 2; hydrogen peroxide; HEPES; 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; PBS; phosphate buffered saline, Amplex Red,; N; -acetyl-3,7-dihydroxyphenoxazineCytochrome c; Cardiolipin; Tyrosine; Cardiolipin hydroperoxide; Peroxidase
Synthesis and characterization of degradable multivalent cationic lipids with disulfide-bond spacers for gene delivery by Rahau S. Shirazi; Kai K. Ewert; Cecilia Leal; Ramsey N. Majzoub; Nathan F. Bouxsein; Cyrus R. Safinya (pp. 2156-2166).
Gene therapy provides powerful new approaches to curing a large variety of diseases, which are being explored in ongoing worldwide clinical trials. To overcome the limitations of viral gene delivery systems, synthetic nonviral vectors such as cationic liposomes (CLs) are desirable. However, improvements of their efficiency at reduced toxicity and a better understanding of their mechanism of action are required. We present the efficient synthesis of a series of degradable multivalent cationic lipids (CMVLn, n=2 to 5) containing a disulfide bond spacer between headgroup and lipophilic tails. This spacer is designed to be cleaved in the reducing milieu of the cytoplasm and thus decrease lipid toxicity. Small angle X-ray scattering demonstrates that the initially formed lamellar phase of CMVLn–DNA complexes completely disappears when reducing agents such as DTT or the biologically relevant reducing peptide glutathione are added to mimic the intracellular milieu. The CMVLs (n=3 to 5) exhibit reduced cytotoxicity and transfect mammalian cells with efficiencies comparable to those of highly efficient non-degradable analogs and benchmark commercial reagents such as Lipofectamine 2000. Thus, our results demonstrate that degradable disulfide spacers may be used to reduce the cytotoxicity of synthetic nonviral gene delivery carriers without compromising their transfection efficiency.► Synthesis of a series of degradable multivalent cationic lipids (CMVLs), 2+ to 5+ ► Disulfide spacer between head and tails is reductively cleaved in cytoplasm ► Triggered disintegration of disulfide spacers significantly reduces cytotoxicity ► Reductive cleavage disassembles CMVLn–DNA complexes ► CMVLs (3+ to 5+) transfect as efficiently as benchmark reagents
Keywords: Abbreviations; Boc; tert; -butoxycarbonyl; CL; cationic liposome; DEAD; diethylazodicarboxylate; DIEA; N; ,; N; -diisopropylethylamine; DLS; dynamic light scattering; DOB; 3,4-di(oleyloxy)benzoic acid; DOPC; 1,2-dioleoyl-; sn; -glycero-3-phosphatidylcholine; DOTAP; 2,3-dioleoyloxypropyltrimethylammonium chloride; DTT; dithiothreitol; EtBr; ethidium bromide; GSH; glutathione; L/D; cationic lipid/DNA weight ratio; NA; nucleic acid; NL; neutral lipid; RLU; relative light units; SAXS; small-angle X-ray scattering; TBTU; 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate; TE; transfection efficiency; TFA; trifluoroacetic acid; TLC; thin-layer chromatography; TL/D; total lipid/DNA weight ratio; Φ; i; mole fraction of lipid i in the lipid mixture; σ; M; membrane charge densityGene delivery; Cationic lipid; Cationic liposome; Nonviral
Conformational changes of β2-human glycoprotein I and lipid order in lipid–protein complexes by Mariana Paolorossi; Guillermo G. Montich (pp. 2167-2177).
We studied the conformation of β2-human glycoprotein (β2GPI) in solution and bound to the anionic lipids palmitoyl oleoyl phosphatidylglycerol (POPG), dimiristoyl phosphatidylglycerol (DMPG) and dipalmitoyl phosphatidylglycerol (DPPG) as a function of the temperature. We used the infrared amide I′ band to study the protein conformation, and the position of the antisymmetric stretching band of the methylene groups in the lipid hydrocarbon chains to study the lipid order. Lipid–protein complexes were studied in media of low and high ionic strengths. In solution, β2GPI displayed a conformational pre-transition in the range 47–50°C, characterized by a shift in the band of β secondary structure, previous to the main unfolding at 64°C. When the protein was bound to the anionic lipid membranes at 25°C, a similar shift as in the pre-transition in solution was observed, together with an increase in the band corresponding to α-helix secondary structure. Lipid–protein complexes formed large aggregates within the temperature range 10≅60°C. At temperatures above the protein unfolding, the complexes were disrupted to yield vesicles with bound protein. This finding indicated that the native fold was required for the formation of the lipid–protein aggregates. Cycles of heating and cooling showed hysteresis in the formation of aggregates.► Beta2 human glycoprotein I–anionic lipid complexes were studied by infrared spectroscopy. ► The proportion of alpha helix increased in the lipid-bound protein. ► The lipid order increased in the lipid–protein complexes. ► Complexes were disrupted when the protein was unfolded by increasing the temperature.
Keywords: Abbreviations; β2GPI; β2-human glycoprotein I; DPPG; 1-palmitoyl-2-palmitoyl-; sn; -glycero-3-phosphoglycerol; DMPG; 1-myristoyl-2-myristoyl-; sn; -glycero-3-phosphoglycerol; POPG; 1-palmitoyl-2-oleoyl-; sn; -glycero-3-phosphoglycerol; FTIR; Fourier transform infrared; FSD; Fourier self deconvolutionβ2-Human glycoprotein I; Lipid membrane; FTIR; Protein conformation; Lipid–protein complex; Aggregation
Effect of E1(64–81) hepatitis G peptide on the in vitro interaction of HIV-1 fusion peptide with membrane models by Sanchez-Martin Maria Jesús Sánchez-Martín; M. Antònia Busquets; Victoria Girona; Isabel Haro; M. Asunción Alsina; Montserrat Pujol (pp. 2178-2188).
One way to gain information about the fusogenic potential of virus-derived synthetic peptides is to examine their interfacial properties and subsequently to study them in monolayers and bilayers. Here, we characterize the physicochemical surface properties of the peptide E1(64–81), whose sequence is AQLVGELGSLYGPLSVSA. This peptide is derived from the E1 structural protein of GBV-C/HGV which was previously shown to inhibit leakage of vesicular contents caused by the HIV-1 fusion peptide (HIV-1 FP). Mixed isotherms of E1(64–81) and HIV-1 FP were obtained and their Brewster angle microscopy (BAM) and atomic force microscopy (AFM) images showed that the peptide mixture forms a different structure that is not present in the pure peptide images. Studies with lipid monolayers (1,2-dimyristoyl- sn-glycero-3-[phospho- rac-(1-glycerol)] (DMPG) and 1,2-dipalmitoyl- sn-glycero-3-phospho- rac-(1-glycerol) (DPPG)) show that both peptides interact with all the lipids assayed but the effect that HIV-1 FP has on the monolayers is reduced in the presence of E1(64–81). Moreover, differential scanning calorimetry (DSC) experiments show the capacity of HIV-1 FP to modify the properties of the bilayer structure and the capacity of E1(64–81) to inhibit these modifications. Our results indicate that E1(64–81) interacts with HIV-1 FP to form a new structure, and that this may be the cause of the previously observed inhibition of the activity of HIV-1 FP by E1(64–81).Display Omitted► Synthetic peptide E1(64–81) of Hepatitis G virus as possible inhibitor of HIV entry. ► E1(64–81) and HIV-1 fusion peptide (FP) forms a new specie when incubated together. ► E1(64–81) inhibits the action of HIV-1 FP on phospholipid monolayers. ► E1(64–81) reduces the interaction of HIV-1 FP with bilayers.
Keywords: Hepatitis GB virus C; Synthetic peptide; Lipid monolayer; Compression isotherm; HIV-1 FP; Bilayer
Analysis of AcrB and AcrB/DARPin ligand complexes by LILBID MS by Brandstatter Lorenz Brandstätter; Lucie Sokolova; Thomas Eicher; Markus A. Seeger; Christophe Briand; Hi-jea Cha; Mihaela Cernescu; Jürgen Bohnert; Winfried V. Kern; Bernd Brutschy; Klaas M. Pos (pp. 2189-2196).
The AcrA/AcrB/TolC complex is responsible for intrinsic multidrug resistance (MDR) in Escherichia coli. Together with the periplasmic adaptor protein AcrA and the outer membrane channel TolC, the inner membrane component AcrB forms an efflux complex that spans both the inner and outer membrane and bridges the periplasm of the Gram-negative cell. Within the entire tripartite complex, homotrimeric AcrB plays a central role in energy transduction and substrate selection. In vitro selected designed ankyrin repeat proteins (DARPin) that specifically bind to the periplasmic domain of AcrB were shown to ameliorate diffraction resolution of AcrB/DARPin protein co-crystals (G. Sennhauser, P. Amstutz, C. Briand, O. Storchenegger, M.G. Grutter, Drug export pathway of multidrug exporter AcrB revealed by DARPin inhibitors, PLoS Biol 5 (2007) e7). Structural analysis by X-ray crystallography revealed that 2 DARPin molecules were bound to the trimeric AcrB wildtype protein in the crystal, whereas the V612F and G616N AcrB variant crystal structures show 3 DARPin molecules bound to the trimer. These specific stoichiometric differences were analyzed in solution via densitometry after microchannel electrophoresis, analytical ultracentrifugation and via laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS). Using the latter technology, we investigated the gradual disassembly of the AcrB trimer and bound DARPin ligands in dependence on laser intensity in solution. At low laser intensity, the release of the detergent molecule micelle from the AcrB/DARPin complex was observed. By increasing laser intensity, dimeric and monomeric AcrB species with bound DARPin molecules were detected showing the high affinity binding of DARPin to monomeric AcrB species. High laser intensity LILBID MS experiments indicated a spectral shift of the monomeric AcrB peak of 3.1kDa, representing a low molecular weight ligand in all detergent-solubilized AcrB samples and in the AcrB crystal. The identity of this ligand was further investigated using phospholipid analysis of purified AcrB and AcrB variant samples, and indicated the presence of phosphatidylethanolamine and possibly cardiolipin, both constituents of the Escherichia coli membrane.► 3 different AcrB variants with DARPins were analyzed via LILBID-MS, SEC and AUC. ► AcrB/DARPin complex stoichiometry differs depending on the AcrB variant used. ► AcrB trimerization is due to strong monomer interaction via the periplasmic loop. ► DARPin binding is stronger than the interaction between the AcrB monomers. ► A 3.1kDa mass attached to the AcrB monomer might represent detergent and/or lipids.
Keywords: AcrB; DARPins; Multidrug efflux; Membrane protein complex; Complex formation; LILBID MS
Structural basis for the enhanced activity of cyclic antimicrobial peptides: The case of BPC194 by Jacek T. Mika; Gemma Moiset; Anna D. Cirac; Lidia Feliu; Bardaji Eduard Bardají; Marta Planas; Durba Sengupta; Siewert J. Marrink; Bert Poolman (pp. 2197-2205).
We report the molecular basis for the differences in activity of cyclic and linear antimicrobial peptides. We iteratively performed atomistic molecular dynamics simulations and biophysical measurements to probe the interaction of a cyclic antimicrobial peptide and its inactive linear analogue with model membranes. We establish that, relative to the linear peptide, the cyclic one binds stronger to negatively charged membranes. We show that only the cyclic peptide folds at the membrane interface and adopts a β-sheet structure characterised by two turns. Subsequently, the cyclic peptide penetrates deeper into the bilayer while the linear peptide remains essentially at the surface. Finally, based on our comparative study, we propose a model characterising the mode of action of cyclic antimicrobial peptides. The results provide a chemical rationale for enhanced activity in certain cyclic antimicrobial peptides and can be used as a guideline for design of novel antimicrobial peptides.Display Omitted► Molecular basis of activity of cyclic antimicrobial peptides. ► Differential partitioning and folding dynamics of active and inactive analogous peptides. ► Crucial arrangement of lysine residues speculated to be implicated in function.
Keywords: Antimicrobial peptide; Peptide-membrane interaction; Molecular dynamics; Peptide folding; Structure–function of cyclic peptide
Folding studies of purified LamB protein, the maltoporin from the Escherichia coli outer membrane: Trimer dissociation can be separated from unfolding by Valerie Baldwin; Mandeep Bhatia; Mary Luckey (pp. 2206-2213).
The folding mechanisms for β-barrel membrane proteins present unique challenges because acquisition of both secondary and tertiary structure is coupled with insertion into the bilayer. For the porins in Escherichia coli outer membrane, the assembly pathway also includes association into homotrimers. We study the folding pathway for purified LamB protein in detergent and observe extreme hysteresis in unfolding and refolding, as indicated by the shift in intrinsic fluorescence. The strong hysteresis is not seen in unfolding and refolding a mutant LamB protein lacking the disulfide bond, as it unfolds at much lower denaturant concentrations than wild type LamB protein. The disulfide bond is proposed to stabilize the structure of LamB protein by clasping together the two sides of Loop 1 as it lines the inner cavity of the barrel. In addition we find that low pH promotes dissociation of the LamB trimer to folded monomers, which run at about one third the size of the native trimer during SDS PAGE and are much more resistant to trypsin than the unfolded protein. We postulate the loss at low pH of two salt bridges between Loop 2 of the neighboring subunit and the inner wall of the monomer barrel destabilizes the quaternary structure.► Unfolding and refolding LamB protein in detergent exhibits extreme hysteresis. ► Without its disulfide bond, LamB protein unfolds in low denaturant concentrations. ► The disulfide bond clasps the sides of L1 at the inner wall of the β-barrel. ► Low pH dissociates the LamB trimer to folded monomers, observable on SDS PAGE. ► Low pH disrupts salt bridges between L2 of the adjacent subunit and the β- barrel.
Keywords: Abbreviations; octylPOE; n-octyl (polydisperse)oligo-oxyethylene; SDS; sodium dodecyl sulfate; DM; dodecylmaltoside; GdmCl; guanidinium chloride; DTT; dithiothreitol; Hepes; N-2-Hydroxypiperazine-N-ethanesulfonic acid; CD; circular dichroism; PAGE; polyacrylamide gel electrophoresis; DMPC; dimyristoylphosphatidylcholineFolding β-barrel protein; Outer membrane; LamB protein; Maltoporin; Disulfide bond; Oligomerization
A minimal isoform of the TMEM16A protein associated with chloride channel activity by Loretta Ferrera; Paolo Scudieri; Elvira Sondo; Antonella Caputo; Emanuela Caci; Olga Zegarra-Moran; Roberto Ravazzolo; Luis J.V. Galietta (pp. 2214-2223).
TMEM16A protein, also known as anoctamin-1, has been recently identified as an essential component of Ca2+-activated Cl− channels. We previously reported the existence of different TMEM16A isoforms generated by alternative splicing. In the present study, we have determined the functional properties of a minimal TMEM16A protein. This isoform, called TMEM16A( 0), has a significantly shortened amino-terminus and lacks three alternative segments localized in the intracellular regions of the protein (total length: 840 amino acids). TMEM16A( 0) expression is associated with Ca2+-activated Cl− channel activity as measured by three different functional assays based on the halide-sensitive yellow fluorescent protein, short-circuit current recordings, and patch-clamp technique. However, compared to a longer isoform, TMEM16( abc) (total length: 982 amino acids), TMEM16A( 0) completely lacks voltage-dependent activation. Furthermore, TMEM16A( 0) and TMEM16A( abc) have similar but not identical responses to extracellular anion replacement, thus suggesting a difference in ion selectivity and conductance. Our results indicate that TMEM16A( 0) has the basic domains required for anion transport and Ca2+-sensitivity. However, the absence of alternative segments, which are present in more complex isoforms of TMEM16A, modifies the channel gating and ion transport ability.► Two isoforms of the TMEM16A chloride channel were analyzed by functional assays.► Both isoforms are activated by calcium and transport anions.► The shorter isoform, TMEM16A( 0), is devoid of voltage-dependence.► Alternative splicing modifies the properties of TMEM16A-associated ion channels.
Keywords: Abbreviations; CaCC; Ca; 2+; -activated Cl; −; channel; YFP; yellow fluorescent proteinChloride channel; Alternative splicing; Intracellular calcium; Patch-clamp
NMR structural study of the intracellular loop 3 of the serotonin 5-HT1A receptor and its interaction with calmodulin by Angela Shuyi Chen; Young Mee Kim; Shovanlal Gayen; Qiwei Huang; Manfred Raida; CongBao Kang (pp. 2224-2232).
The serotonin (5-HT1A) receptor, a G-protein-coupled receptor (GPCR), plays important roles in serotonergic signaling in the central nervous system. The third intracellular loop (ICL3) of the 5-HT1A receptor has been shown to be important for the regulation of this receptor through interactions with proteins such as G-proteins and calmodulin. In this study, the ICL3 of 5-HT1A receptor was expressed in E. coli and purified. Gel filtration and mass spectrometry were used to confirm the molecular weight of the purified ICL3. Secondary structure analysis using circular dichroism (CD) demonstrated the presence of α-helical structures. Backbone assignment of ICL3 was achieved using three-dimensional experiments. A chemical shift index and Talos+ analysis showed that residues E326 to R339 form α-helical structure. Residues G256 to S269 of ICL3 were shown to be a novel region that has a molecular interaction with calmodulin in titration assays. Peptide derived from the ICL3 containing residues from G256 to S269 also showed molecular interaction with calmodulin.► The intracellular Loop 3 (ICL3) of the Serotonin 5-HT2A receptor was purified. ► Backbone resonance assignment showed the presence of an alpha-helical structure. ► A new calmodulin binding site was identified. ► Both N and C terminus of calmodulin are affected by its interaction with ICL3.
Keywords: 5-HT; 1A; receptor; GPCR; NMR spectroscopy; Membrane protein; Calmodulin; Intracellular loop 3
Spectroscopic analysis of the intrinsic chromophores within small multidrug resistance protein SugE by Denice C. Bay; Raymond J. Turner (pp. 2233-2244).
Small multidrug resistance (SMR) protein family member, SugE, is an integral inner membrane protein that confers host resistance to antiseptic quaternary cation compounds (QCC). SugE studies generally focus on its resistance to limited substrates in comparison to SMR protein EmrE. This study examines the conformational characteristics of SugE protein in two detergents, sodium dodecyl sulphate (SDS) and dodecyl maltoside (DDM), commonly used to study SMR proteins. The influence of cetylpyridinium (CTP) and cetrimide (CET) using SugE aromatic residues (4W, 2Y, 1F) as intrinsic spectroscopic probes was also determined. Organically extracted detergent solubilized Escherichia coli SugE protein was examined by SDS-Tricine PAGE and various spectroscopic techniques. SDS-Tricine PAGE analysis of SugE in either detergent demonstrates the protein predominates as a monomer but also dimerizes in SDS. Far-UV region circular dichroism (CD) analysis determined that the overall α-helix content SugE in SDS and DDM was almost identical and unaltered by QCC. Near-UV region CD, fluorescence, and second-derivative ultraviolet absorption (SDUV) indicated that only DDM-SugE promoted hydrophobic environments for its Trp and Tyr residues that were perturbed by QCC addition. This study identified that only the tertiary structure of SugE protein in DDM is altered by QCC.► We examine SugE intrinsic aromatic amino acid residue conformations. ► We characterize SugE conformation in two detergents SDS and DDM. ► We determine that the alpha-helix content of SugE is unchanged by detergent or antiseptic.c ► We examine that antiseptics alter SugE tertiary conformation in DDM only. ► We identify that each antiseptic uniquely alters DDM-SugE aromatic residue arrangements.
Keywords: Small multidrug resistance protein (SMR); Suppressor of GroEL mutations (SugE); Quaternary cation compound (QCC); Cetrimide; Cetylpyridinium; Quaternary ammonium compound (QAC)
The effect of oxycholesterols on thermo-induced membrane dynamics by Mun'delanji C. Vestergaard; Tsuyoshi Yoda; Tsutomu Hamada; Yoko Akazawa (Ogawa); Yasukazu Yoshida; Masahiro Takagi (pp. 2245-2251).
The effect of temperature change(s) on the dynamics of giant unilamellar vesicles containing oxidized and non-oxidized cholesterol was investigated and characterized. We have demonstrated that (i) major cholesterol auto-oxidation products, 7β-hydroxycholesterol (7β) and 7-ketocholesterol (7keto), rendered vesicles more responsive to temperature changes; (ii) 7keto imparted greater thermo-induced membrane dynamics than 7β; (iii) 7β and 7keto vesicles synergistically were more thermo-responsive than the individual oxysterols; (iv) the thermo-responsiveness of 7keto-containing vesicles was equivalent to that of 25 hydroxycholesterol (25OH)-containing vesicles; and (v) we have characterized the observed membrane dynamics. The results provide a new plausible mechanism: oxidative-stressed membranes in conjunction with temperature change induce membrane dynamics. These findings improve the mechanisms reported previously that attributed the induced dynamics solely to membrane oxidation.► We demonstrate that oxy-cholesterol-containing lipid vesicles were more sensitive to temperature. ► We show that keto functional group rendered lipid vesicles more thermo-sensitive than OH group at carbon 7. ► We show that OH group at C 25 has the same effect as the keto group at C 7. ► Characterization of the membrane dynamics provide a new insight into oxidative-stressed membranes. ► We show that temperature change in conjunction with membrane oxidation induces membrane instability.
Keywords: Lipid vesicle; Cholesterol; Oxidized cholesterol model membrane; Oxidative stress; Thermo-response
Selective permeabilization of lipid membranes by photodynamic action via formation of hydrophobic defects or pre-pores by Elena A. Kotova; Alexey V. Kuzevanov; Alina A. Pashkovskaya; Yuri N. Antonenko (pp. 2252-2257).
To gain insight into mechanisms of photodynamic modification of biological membranes, we studied an impact of visible light in combination with a photosensitizer on translocation of various substances across artificial (vesicular and planar) bilayer lipid membranes (BLMs). Along with induction of carboxyfluorescein leakage from liposomes, pronounced stimulation of lipid flip-flop between the two monolayers was found after photosensitization, both processes being prevented by the singlet oxygen quencher sodium azide. On the contrary, no enhancement of potassium chloride efflux from liposomes was detected by conductometry under these conditions. Illumination of planar BLMs in the presence of a photosensitizer led to a marked increase in membrane permeability to amphiphilic 2- n-octylmalonic acid, but practically no change in the permeability to ammonia, which agreed with selective character of the photosensitized leakage of fluorescent dyes from liposomes (Pashkovskaya et al., Langmuir, 2010). Thus, the effect on transbilayer movement of molecules elicited by the photodynamic treatment substantially depended on the kind of translocated species, in particular, on their lipophilicity. Based on similarity with results of previous electroporation studies, we hypothesized about photodynamic induction of “pre-pores” or “hydrophobic defects” permeable to amphiphilic compounds and less permeable to hydrophilic substances and inorganic ions.► Photosensitization induces lipid flip-flop and carboxyfluorescein leakage in unsaturated liposomes via singlet oxygen attack. ► Photodynamic treatment does not stimulate potassium chloride efflux from liposomes. ► Photodynamically induced membrane permeability depends on lipophilicity of permeating compounds. ► Oxidative permeabilization of membranes involves formation of hydrophobic pores.
Keywords: Abbreviations; BLM; bilayer lipid membrane; DPhPC; diphytanoylphosphatidylcholine; eggPC; egg yolk phosphatidylcholine; Chol; cholesterol; TTFB; tetrachlorotrifluoromethylbenzeimidazole; α,α-DC11; 2-; n; -octylmalonic acid; CF; 5(6)-carboxyfluorescein; pyPC; 1-lauroyl-2-(1′pyrenebutyroyl)-sn-glycero-3-phosphocholine; AlPcS; 3; aluminum trisulfophthalocyanine; USL; unstirred layerLiposome; Leakage; Bilayer lipid membrane; Pore; Photosensitizer; Lipid peroxidation
Influence of the arrangement and secondary structure of melittin peptides on the formation and stability of toroidal pores by Sheeba J. Irudayam; Max L. Berkowitz (pp. 2258-2266).
Melittin interactions with lipid bilayers and melittin formed pores are extensively studied to understand the mechanism of the toroidal pore formation. Early experimental studies suggested that melittin peptide molecules are anchored by their positively charged residues located next to the C-terminus to only one leaflet of the lipid bilayer (asymmetric arrangement). However, the recent non-linear spectroscopic experiment suggests a symmetric arrangement of the peptides with the C-terminus of the peptides anchored to both bilayers. Therefore, we present here a computational study that compares the effect of symmetric and asymmetric arrangements of melittin peptides in the toroidal pore formation. We also investigate the role of the peptide secondary structure during the pore formation. Two sets of the symmetric and asymmetric pores are prepared, one with a helical peptide from the crystal structure and the other set with a less helical peptide. We observe a stable toroidal pore being formed only in the system with a symmetric arrangement of the less helical peptides. Based on the simulation results we propose that the symmetric arrangement of the peptides might be more favorable than the asymmetric arrangement, and that the helical secondary structure is not a prerequisite for the formation of the toroidal pore.► Simulated symmetrically and asymmetrically arranged melittins in a POPC bilayer pore. ► For the first time observed creation of the fully toroidal pore in symmetric arrangement. ► Observed that helicity is not a prerequisite for the toroidal pore creation. ► Investigated the role of melittin charge distribution promoting toroidal pore creation. ► Proposed that the symmetrical arrangement of melittin peptides is more favorable for the toroidal pore creation.
Keywords: Antimicrobial peptide; Molecular dynamics simulation; Toroidal pore; Melittin
Cholesterol attenuates and prevents bilayer damage and breakdown in lipoperoxidized model membranes. A spin labeling EPR study by Francesco M. Megli; Elena Conte; Takashi Ishikawa (pp. 2267-2274).
The stabilizing effect of cholesterol on oxidized membranes has been studied in planar phospholipid bilayers and multilamellar 1-palmitoyl-2-linoleoyl-phosphatidylcholine vesicles also containing either 1-palmitoyl-2-glutaroyl-phosphatidylcholine or 1-palmitoyl-2-(13-hydroxy-9,11-octadecanedienoyl)-phosphatidylcholine oxidized phosphatidylcholine in variable ratio. Lipid peroxidation-dependent membrane alterations in the absence and in the presence of cholesterol were analyzed using Electron Paramagnetic Resonance spectroscopy of the model membranes spin labelled with either cholestane spin label (3-DC) or phosphatidylcholine spin label (5-DSPC). Cholesterol, added to lipid mixtures up to 40% final molar ratio, decreased the inner bilayer disorder as compared to cholesterol-free membranes and strongly reduced bilayer alterations brought about by the two oxidized phosphatidylcholine species. Furthermore, Sepharose 4B gel-chromatography and cryo electron microscopy of aqueous suspensions of the lipid mixtures clearly showed that cholesterol is able to counteract the micelle forming tendency of pure 1-palmitoyl-2-glutaroyl-phosphatidylcholine and to sustain multilamellar vesicles formation. It is concluded that membrane cholesterol may exert a beneficial and protective role against bilayer damage caused by oxidized phospholipids formation following reactive oxygen species attack to biomembranes.Display Omitted► 40% Cholesterol in lipoperoxidized planar membranes prevents fatty acid disorienting. ► 40% Cholesterol in oxidized phospholipid vesicles counteracts membrane breakdown. ► Cholesterol maintains bilayer ordering and preserves membrane integrity in lipoperoxidation.
Keywords: Abbreviations; C; cholesterol; 3-DC; 3-doxyl-cholestane spin label; 5-DSPC; 2-(5-doxylstearoyl)- phosphatidylcholine; EPR (ESR); Electron Paramagnetic Resonance; lyso-PC; 2-lyso-phosphatidylcholine; HOPLPC; 1-palmitoyl-2-(13-hydroxy-9,11-octadecanedienoyl)-phosphatidylcholine; MLV; multilamellar vesicles; OXPC; oxidized phosphatidylcholine; PC; phosphatidylcholine; PGPC; 1-palmitoyl-2-glutaroyl-phosphatidylcholine; PLPC; 1-palmitoyl-2-linoleoyl-phosphatidylcholine; ROS; reactive oxygen species; SPB; supported planar bilayers; SUV; small unilamellar vesiclesPhospholipid bilayer; Cholesterol; Lipoperoxidation; EPR-Spin labeling; Bilayer disordering; EPR spectral anisotropy
The behavior of sea anemone actinoporins at the water–membrane interface by Garcia-Ortega Lucía García-Ortega; Jorge Alegre-Cebollada; Garcia-Linares Sara García-Linares; Marta Bruix; Martinez-del-Pozo Álvaro Martínez-del-Pozo; José G. Gavilanes (pp. 2275-2288).
Actinoporins constitute a group of small and basic α-pore forming toxins produced by sea anemones. They display high sequence identity and appear as multigene families. They show a singular behaviour at the water–membrane interface: In aqueous solution, actinoporins remain stably folded but, upon interaction with lipid bilayers, become integral membrane structures. These membranes contain sphingomyelin, display phase coexistence, or both. The water soluble structures of the actinoporins equinatoxin II (EqtII) and sticholysin II (StnII) are known in detail. The crystalline structure of a fragaceatoxin C (FraC) nonamer has been also determined. The three proteins fold as a β-sandwich motif flanked by two α-helices, one of them at the N-terminal end. Four regions seem to be especially important: A cluster of aromatic residues, a phosphocholine binding site, an array of basic amino acids, and the N-terminal α-helix. Initial binding of the soluble monomers to the membrane is accomplished by the cluster of aromatic amino acids, the array of basic residues, and the phosphocholine binding site. Then, the N-terminal α-helix detaches from the β-sandwich, extends, and lies parallel to the membrane. Simultaneously, oligomerization occurs. Finally, the extended N-terminal α-helix penetrates the membrane to build a toroidal pore. This model has been however recently challenged by the cryo-EM reconstruction of FraC bound to phospholipid vesicles. Actinoporins structural fold appears across all eukaryotic kingdoms in other functionally unrelated proteins. Many of these proteins neither bind to lipid membranes nor induce cell lysis. Finally, studies focusing on the therapeutic potential of actinoporins also abound.Display Omitted► Actinoporins constitute a group of small and basic α-pore forming toxins produced by sea anemones. ► In water remain stably folded but upon interaction with membranes become integral membrane proteins. ► These membranes contain sphingomyelin, display phase coexistence, or both. ► Initial binding of the monomers to the membrane is accomplished by its structural β-sandwich core. ► Protein oligomerizes, N-terminal α-helix detaches, extends and builds the pore.
Keywords: Abbreviations; Avt; actinoporins from; Actineria villosa; ALP; actinoporin-like protein; ATR; attenuated total reflection; Bc2; actinoporin from; Bunodosoma caissarum; CD; circular dichroism; Chol; cholesterol; DMPC; dimyristoylphosphatidylcholine; DOPC; dioleylphosphatidylcholine; DPC; dodecylphosphocholine; DrI; ALP from; Danio rerio; EM; electron microscopy; Ent; actinoporin from; Entacmea quadricolor; Eqt; equinatoxin; FTIR; Fourier transform infrared spectroscopy, Fra C, actinoporin from; Actinia fragacea; GUV; giant unilamellar vesicles; ITC; isothermal titration calorimetry; NLP; necrosis and ethylene-inducing peptide 1 (Nep1)-like protein; NMR; nuclear magnetic resonance; PE; phosphatidylethanolamine; PFT; pore forming toxin; PpBP; ALP from; Physcomitrella patens; Pstx; actinoporins from; Phyllodiscus semoni; SM; sphingomyelin; SPR; surface plasmon resonance; Stn; sticholysin; TFE; trifluoroethanolActinoporin; Equinatoxin; Sticholysin; Membrane-pore; Pore-forming-toxin
Towards understanding the Tat translocation mechanism through structural and biophysical studies of the amphipathic region of TatA from Escherichia coli by Catherine S. Chan; Evan F. Haney; Hans J. Vogel; Raymond J. Turner (pp. 2289-2296).
The twin-arginine translocase (Tat) system is used by many bacteria and plants to move folded proteins across the cytoplasmic or thylakoid membrane. In most bacteria, the TatA protein is believed to form a defined pore in the membrane through homo-oligomerization with other TatA protomers. The predicted secondary structure of TatA includes a transmembrane helix, an amphipathic helix, and an unstructured C-terminal region. Here biophysical and structural investigations were performed on a synthetic peptide representing the amphipathic region of TatA (residues 22 to 44, abbreviated TatAH2). The C-terminal region of TatA (residues 44–89) was previously shown to be accessible from both the cytoplasmic and periplasmic sides of the membrane only when the membrane potential was intact, suggesting dependence of its topology on an energized membrane (Chan et al. 2007 Biochemistry 46: 7396–404). Such observation suggests that the TatAH2 region would have unique lipid interactions that may be related to the function of TatA during translocation and thus warranted further investigations. NMR and CD spectroscopy of TatAH2 show that it adopts a predominantly helical structure in a membrane environment while remaining unstructured in aqueous solution. Differential scanning calorimetry studies also reveal that TatAH2 interacts with DPPG lipids but not with DPPC, suggesting that negatively charged phospholipid head groups contribute to the membrane interactions with TatA.► Helix-2 of TatA is predominantly helical in membrane environments. ► Helix-2 of TatA interacts with negatively charged lipids. ► Solution structure of the TatA helix-2 peptide shows it forms an amphipathic helix.
Keywords: Abbreviations; Tat; twin-arginine translocase; TatAH2; TatA helix-2 peptide (residues 22–44 of full-length sequence); NMR; nuclear magnetic resonance; CD; circular dichroism; DSC; differential scanning calorimetry; DPPG; 1,2-dipalmitoyl-; sn; -glycero-3-phospho-(1′-; rac; -glycerol); DPPC; 1,2-dipalmitoyl-; sn; -glycero-3-phosphocholine; DPC; dodecylphosphocholine; eggPC; egg derived phosphatidylcholine; eggPG; egg derived phosphatidylglycerol; eggPE; egg derived phosphatidylethanolamine; SDS; sodium dodecyl sulfate; SUV; small unilamellar vesicles; LUV; large unilamellar vesiclesTatA; Twin-arginine translocation; NMR solution structure; Amphipathic peptide
Investigating the cationic side chains of the antimicrobial peptide tritrpticin: Hydrogen bonding properties govern its membrane-disruptive activities by Leonard T. Nguyen; Leonie de Boer; Sebastian A.J. Zaat; Hans J. Vogel (pp. 2297-2303).
The positively charged side chains of cationic antimicrobial peptides are generally thought to provide the initial long-range electrostatic attractive forces that guide them towards the negatively charged bacterial membranes. Peptide analogs were designed to examine the role of the four Arg side chains in the cathelicidin peptide tritrpticin (VRRFPWWWPFLRR). The analogs include several noncoded Arg and Lys derivatives that offer small variations in side chain length and methylation state. The peptides were tested for bactericidal and hemolytic activities, and their membrane insertion and permeabilization properties were characterized by leakage assays and fluorescence spectroscopy. A net charge of +5 for most of the analogs maintains their high antimicrobial activity and directs them towards preferential insertion into model bacterial membrane systems with a similar extent of burial of the Trp side chains. However the peptides exhibit significant functional differences. Analogs with methylated cationic side chains cause lower levels of membrane leakage and are associated with lower hemolytic activities, making them potentially attractive pharmaceutical candidates. Analogs containing the Arg guanidinium groups cause more membrane disruption than those containing the Lys amino groups. Peptides in the latter group with shorter side chains have increased membrane activity and conversely, elongating the Arg residue causes slightly higher membrane activity. Altogether, the potential for strong hydrogen bonding between the four positive Arg side chains with the phospholipid head groups seems to be a determinant for the membrane disruptive properties of tritrpticin and many related cationic antimicrobial peptides.► Novel tritrpticin analogs were designed to study the role of its Arg residues. ► Decreasing H-bond capability for the cationic residues reduces membrane disruption. ► Regardless of membrane activity, the analogs retain high antimicrobial activity.
Keywords: Abbreviations; EC; 50; effective concentration for 50% hemolysis; ePC; egg-yolk phosphatidylcholine; ePG; egg-yolk phosphatidylglycerol; LC; 99.9; lethal concentration for 99.9% killing; LUV; large unilamellar vesicleTritrpticin; Antimicrobial peptide; Cathelicidin; Arginine; Membrane-active peptide; Hydrogen bonding
Plant actin controls membrane permeability by Petra Hohenberger; Christian Eing; Ralf Straessner; Steffen Durst; Wolfgang Frey; Peter Nick (pp. 2304-2312).
The biological effects of electric pulses with low rise time, high field strength, and durations in the nanosecond range (nsPEFs) have attracted considerable biotechnological and medical interest. However, the cellular mechanisms causing membrane permeabilization by nanosecond pulsed electric fields are still far from being understood. We investigated the role of actin filaments for membrane permeability in plant cells using cell lines where different degrees of actin bundling had been introduced by genetic engineering. We demonstrate that stabilization of actin increases the stability of the plasma membrane against electric permeabilization recorded by penetration of Trypan Blue into the cytoplasm. By use of a cell line expressing the actin bundling WLIM domain under control of an inducible promotor we can activate membrane stabilization by the glucocorticoid analog dexamethasone. By total internal reflection fluorescence microscopy we can visualize a subset of the cytoskeleton that is directly adjacent to the plasma membrane. We conclude that this submembrane cytoskeleton stabilizes the plasma membrane against permeabilization through electric pulses.Display Omitted► Actin bundling stabilizes plant membranes against electropermeabilization. ► Actin filaments and microtubules are physically close to the plasma membrane. ► Model: actin stabilizes and mobilizes internal stores of membrane material.
Keywords: Abbreviations; nsPEFs; nanosecond pulsed electric fields; TIRF; total internal reflection fluorescenceActin; Plant cell; Pulsed electric field; Tobacco BY-2; Total internal reflection microscopy
Influence of detergents on the activity of the ABC transporter LmrA by Nacera Infed; Nils Hanekop; Arnold J.M. Driessen; Sander H.J. Smits; Lutz Schmitt (pp. 2313-2321).
The ABC transporter LmrA from Lactococcus lactis has been intensively studied and a role in multidrug resistance was proposed. Here, we performed a comprehensive detergent screen to analyze the impact of detergents for a successful solubilization, purification and retention of functional properties of this ABC transporter. Our screen revealed the preference of LmrA for zwitterionic detergents. In detergent solution, LmrA purified with FC-16 was highly active with respect to ATPase activity, which could be stimulated by a substrate (rhodamine 123) of LmrA. Both, high ATPase activity and substrate stimulation were not detected for LmrA solubilized in DDM. Interestingly, reconstituted LmrA showed an opposite behavior, with a high basal ATPase activity and stimulation by rhodamine 123 for a DDM-reconstituted, but only low ATPase activity and no substrate stimulation for a FC-16 reconstituted sample.► We investigated the influence of 40 detergents on the ABC transporter LmrA. ► Solubilization efficiency, oligomeric state and functionality were analyzed. ► LmrA displayed a preference for zwitterionic detergents. ► In detergent solution FC-16 was superior, while DDM displayed modest activity. ► In proteoliposomes, DDM-reconstituted LmrA was superior to FC-16.
Keywords: Abbreviations; DDM; dodecyl-β-; d; -maltoside; FC-16; FOS-CHOLINE-16; MDR; multidrug resistance; SEC; size exclusion chromatography; C; 10; E; 5; Pentaethylene glycol monodecyl etherDetergent screen; ABC transporter; Functional reconstitution; ATPase activity
The effect of long-chain bases on polysialic acid-mediated membrane interactions by Teresa Janas; Krzysztof Nowotarski; Tadeusz Janas (pp. 2322-2326).
Negatively-charged polysialic acid (polySia) chains are usually membrane-bound and are often expressed on the surface of neuroinvasive bacterial cells, neural cells, and tumor cells. PolySia can mediate both repulsive and attractive cis interactions between membrane components, and trans interactions between membranes. Positively-charged long-chain bases are widely present in cells, are often localized in membranes and can function as bioactive lipids. Here we use Langmuir monolayer technique, fluorescence spectroscopy and electron microscopy of lipid vesicles to study the role of a simple long-chain base, octadecylamine (ODA), in both cis and trans interactions mediated by polySia in model membranes composed of ODA and dioleoylphospatidycholine (DOPC). When added free to an aqueous solution, polySia increases the collapse pressure of ODA/DOPC monolayers, reduces the effect of ODA on the limiting molecular area, inverses the values of excess area per molecule and of excess free energy of mixing from positive to negative, and induces fusion of ODA/DOPC vesicles. These results suggest that a polySia chain can act as a multi-bridge that mediates cis interactions between different components of a lipid membrane, disrupts membrane aggregates, and mediates trans interactions between lipids in apposing membranes. These observations imply that polySia in cellular systems can act in a similar way.► polySia chains can mediate cis interactions between ODA and DOPC in ODA/DOPC monolayers. ► polySia chains can disrupt ODA aggregates in ODA/DOPC monolayers. ► polySia chains can mediate trans interactions between apposing ODA/DOPC bilayers. ► polySia chain may function as a multi-bridge attracting the components of ODA/DOPC membranes.
Keywords: Electron microscopy; Fluorescence; Liposome; Octadecylamine; Phospholipid monolayer; Polysialic acid
Structural and functional analysis of critical amino acids in TMVI of the NHE1 isoform of the Na+/H+ exchanger by Jennifer Tzeng; Brian L. Lee; Brian D. Sykes; Larry Fliegel (pp. 2327-2335).
The mammalian Na+/H+ exchanger isoform 1 (NHE1) resides on the plasma membrane and exchanges one intracellular H+ for one extracellular Na+. It maintains intracellular pH and regulates cell volume, and cell functions including growth and cell differentiation. Previous structural and functional studies on TMVI revealed several amino acids that are potentially pore lining. We examined these and other critical residues by site-directed mutagenesis substituting Asn227→Ala, Asp, Arg; Ile233→Ala; Leu243→Ala; Glu247→Asp, Gln; Glu248→Asp, Gln. Mutant NHE1 proteins were characterized in AP-1 cells, which do not express endogenous NHE1. All the TMVI critical amino acids were highly sensitive to substitution and changes often lead to a dysfunctional protein. Mutations of Asn227→Ala, Asp, Arg; Ile233→Ala; Leu243→Ala; Glu247→Asp; Glu248→Gln yielded significant reduction in NHE1 activity. Mutants of Asn227 demonstrated defects in protein expression, targeting and activity. Substituting Asn227→Arg and Ile233→Ala decreased the surface localization and expression of NHE1 respectively. The pore lining amino acids Ile233 and Leu243 were both essential for activity. Glu247 was not essential, but the size of the residue at this location was important while the charge on residue Glu248 was more critical to NHE1 function. Limited trypsin digestion on Leu243→Ala and Glu248→Gln revealed that they had increased susceptibility to proteolytic attack, indicating an alteration in protein conformation. Modeling of TMVI with TMXI suggests that these TM segments form part of the critical fold of NHE1 with Ile233 and Leu465 of TMXI forming a critical part of the extracellular facing ion conductance pathway.► Critical amino acids of TMVI of the Na+/H+ exchanger were mutated. ► TM VI critical residues include Asn227, Ile233, Leu243, Glu247 and Glu248. ► Mutant proteins yielded varying defects in activity expression and targeting. ► Modeling of TMVI indicates it forms a critical part of the ion conductance pathway.
Keywords: Abbreviations; BCECF-AM; 2′,7′-bis(2-carboxyethyl)-5(6) carboxyfluorescein-acetoxymethyl ester; DPC; dodecylphosphocholine; HA; hemagglutinin; Intracellular pH; pH; i; MTSET; ([2-(Trimethylammonium)Ethyl]Methanethiosulfonate); NhaA; E. coli Na; +; /H; +; antiporter type A; NHE; Na; +; /H; +; exchanger; NHE1; Na; +; /H; +; exchanger isoform one; NMR; nuclear magnetic resonance; PBS; phosphate buffered saline; TMVI; transmembrane segment VI; TMXI; transmembrane segment XI; WT; wild typeCation coordination; Membrane protein; Na; +; /H; +; exchanger; NhaA; NMR; Site specific mutagenesis