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Jacek Lipkowski - One of the best experts on this subject based on the ideXlab platform.
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effects of amiloride an ion channel blocker on Alamethicin pore formation in negatively charged gold supported phospholipid bilayers a molecular view
Langmuir, 2019Co-Authors: Fatemeh Abbasi, Jay J. Leitch, Julia Alvarezmalmagro, Jacek LipkowskiAbstract:The effects of amiloride on the structure and conductivity of Alamethicin ion pore formation within negatively charged, gold-supported, 1,2-dimyristoyl- sn-glycero-3-phosphocholine/Egg-PG membranes were investigated with the help of electrochemical impedance spectroscopy (EIS), photon polarization modulation-infrared reflection spectroscopy (PM-IRRAS), and atomic force microscopy (AFM). The EIS results indicate that ion conductivity across negatively charged phospholipid bilayers containing Alamethicin decreases by an order of magnitude when amiloride is introduced to the system. Despite the reduction in ion conductivity, the PM-IRRAS data shows that amiloride does not inhibit ion channel formation by Alamethicin peptides. High-resolution AFM images revealed that amiloride enlarges and distorts the shape of Alamethicin ion pores when introduced to the system, indicating that it is inserting itself into the mouth of the Alamethicin pores. This effect is driven by electrostatic interactions between positively charged amiloride molecules and the negative charge on the membrane.
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Mechanisms of Alamethicin ion channel inhibition by amiloride in zwitterionic tethered lipid bilayers
Journal of Electroanalytical Chemistry, 2019Co-Authors: Fatemeh Abbasi, J. Jay Leitch, Robert J. Faragher, Adrian L. Schwan, Jacek LipkowskiAbstract:Abstract The effect of amiloride, an acid-sensing ion channel (ASIC) blocker, on the formation of Alamethicin ion channels in tethered zwitterionic phospholipid bilayers was investigated using electrochemical impedance spectroscopy (EIS) and polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS). EIS measurements show a decrease in conductivity indicating that ion transported across the Alamethicin-rich phospholipid membrane is inhibited by the presence of amiloride. The PM-IRRAS spectra indicate that amiloride has no effect on the secondary structure of the Alamethicin peptide. The IR data suggests that amiloride does not block ion translocation through the Alamethicin ion pore but prevents the insertion of Alamethicin peptides into the biomembrane suppressing the formation of ion channels. The mechanism responsible for amiloride inhibition is explained in terms of the electrostatic interactions between the molecule and membrane surface. Amiloride is positively charged at physiological pH levels invoking a repulsive force on the positive pole of the Alamethicin molecular dipole. These repulsive interactions prevent the insertion of the Alamethicin peptides into the hydrophobic core of the lipid bilayer. The present paper offers a molecular view of the interactions that occur between amiloride, an ion channel inhibitor, with the lipids and ion channel forming peptides in model tBLMs.
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Effects of Amiloride, an Ion Channel Blocker, on Alamethicin Pore Formation in Negatively Charged, Gold-Supported, Phospholipid Bilayers: A Molecular View
2019Co-Authors: Fatemeh Abbasi, Jay J. Leitch, Julia Alvarez-malmagro, Jacek LipkowskiAbstract:The effects of amiloride on the structure and conductivity of Alamethicin ion pore formation within negatively charged, gold-supported, 1,2-dimyristoyl-sn-glycero-3-phosphocholine/Egg-PG membranes were investigated with the help of electrochemical impedance spectroscopy (EIS), photon polarization modulation-infrared reflection spectroscopy (PM-IRRAS), and atomic force microscopy (AFM). The EIS results indicate that ion conductivity across negatively charged phospholipid bilayers containing Alamethicin decreases by an order of magnitude when amiloride is introduced to the system. Despite the reduction in ion conductivity, the PM-IRRAS data shows that amiloride does not inhibit ion channel formation by Alamethicin peptides. High-resolution AFM images revealed that amiloride enlarges and distorts the shape of Alamethicin ion pores when introduced to the system, indicating that it is inserting itself into the mouth of the Alamethicin pores. This effect is driven by electrostatic interactions between positively charged amiloride molecules and the negative charge on the membrane
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pore forming properties of Alamethicin in negatively charged floating bilayer lipid membranes supported on gold electrodes
Langmuir, 2018Co-Authors: Fatemeh Abbasi, Jay J. Leitch, Julia Alvarezmalmagro, Jacek LipkowskiAbstract:Electrochemical impedance spectroscopy (EIS), atomic force microscopy (AFM), and photon polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) were employed to investigate the formation of Alamethicin pores in negatively charged bilayers composed of a mixture of 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) and egg-PG floating at gold (111) electrode surfaces modified by self-assembled monolayers of 1-thio-β-d-glucose (β-Tg). The EIS data showed that the presence of Alamethicin decreases the membrane resistivity by about 1 order of magnitude. PM-IRRAS measurements provided information about the tilt angles of peptide helical axis with respect to the bilayer normal. The small tilt angles obtained for the peptide helical axis prove that the Alamethicin molecules were inserted into the DMPC/egg-PG membranes. The tilt angles decreased when negative potentials were applied, which correlates with the observed decrease in membrane resistivity, indicating that ion pore formation is assisted by the transmembrane potential. Molecular resolution AFM images provided visual evidence that Alamethicin molecules aggregate forming hexagonal porous 2D lattices with periodicities of 2.0 ± 0.2 nm. The pore formation by Alamethicin in the negatively charged membrane was compared with the interaction of this peptide with a bilayer formed by zwitterionic lipids. The comparison of these results showed that Alamethicin preferentially forms ion translocating pores in negatively charged phospholipid membranes.
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pore forming properties of Alamethicin in negatively charged floating bilayer lipid membranes supported on gold electrodes
Langmuir, 2018Co-Authors: Fatemeh Abbasi, Jay J. Leitch, Julia Alvarezmalmagro, Jacek LipkowskiAbstract:Electrochemical impedance spectroscopy (EIS), atomic force microscopy (AFM), and photon polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) were employed to investigate the formation of Alamethicin pores in negatively charged bilayers composed of a mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and egg-PG floating at gold (111) electrode surfaces modified by self-assembled monolayers of 1-thio-β-d-glucose (β-Tg). The EIS data showed that the presence of Alamethicin decreases the membrane resistivity by about 1 order of magnitude. PM-IRRAS measurements provided information about the tilt angles of peptide helical axis with respect to the bilayer normal. The small tilt angles obtained for the peptide helical axis prove that the Alamethicin molecules were inserted into the DMPC/egg-PG membranes. The tilt angles decreased when negative potentials were applied, which correlates with the observed decrease in membrane resistivity, indicating that ion pore formation is as...
Fatemeh Abbasi - One of the best experts on this subject based on the ideXlab platform.
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effects of amiloride an ion channel blocker on Alamethicin pore formation in negatively charged gold supported phospholipid bilayers a molecular view
Langmuir, 2019Co-Authors: Fatemeh Abbasi, Jay J. Leitch, Julia Alvarezmalmagro, Jacek LipkowskiAbstract:The effects of amiloride on the structure and conductivity of Alamethicin ion pore formation within negatively charged, gold-supported, 1,2-dimyristoyl- sn-glycero-3-phosphocholine/Egg-PG membranes were investigated with the help of electrochemical impedance spectroscopy (EIS), photon polarization modulation-infrared reflection spectroscopy (PM-IRRAS), and atomic force microscopy (AFM). The EIS results indicate that ion conductivity across negatively charged phospholipid bilayers containing Alamethicin decreases by an order of magnitude when amiloride is introduced to the system. Despite the reduction in ion conductivity, the PM-IRRAS data shows that amiloride does not inhibit ion channel formation by Alamethicin peptides. High-resolution AFM images revealed that amiloride enlarges and distorts the shape of Alamethicin ion pores when introduced to the system, indicating that it is inserting itself into the mouth of the Alamethicin pores. This effect is driven by electrostatic interactions between positively charged amiloride molecules and the negative charge on the membrane.
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Mechanisms of Alamethicin ion channel inhibition by amiloride in zwitterionic tethered lipid bilayers
Journal of Electroanalytical Chemistry, 2019Co-Authors: Fatemeh Abbasi, J. Jay Leitch, Robert J. Faragher, Adrian L. Schwan, Jacek LipkowskiAbstract:Abstract The effect of amiloride, an acid-sensing ion channel (ASIC) blocker, on the formation of Alamethicin ion channels in tethered zwitterionic phospholipid bilayers was investigated using electrochemical impedance spectroscopy (EIS) and polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS). EIS measurements show a decrease in conductivity indicating that ion transported across the Alamethicin-rich phospholipid membrane is inhibited by the presence of amiloride. The PM-IRRAS spectra indicate that amiloride has no effect on the secondary structure of the Alamethicin peptide. The IR data suggests that amiloride does not block ion translocation through the Alamethicin ion pore but prevents the insertion of Alamethicin peptides into the biomembrane suppressing the formation of ion channels. The mechanism responsible for amiloride inhibition is explained in terms of the electrostatic interactions between the molecule and membrane surface. Amiloride is positively charged at physiological pH levels invoking a repulsive force on the positive pole of the Alamethicin molecular dipole. These repulsive interactions prevent the insertion of the Alamethicin peptides into the hydrophobic core of the lipid bilayer. The present paper offers a molecular view of the interactions that occur between amiloride, an ion channel inhibitor, with the lipids and ion channel forming peptides in model tBLMs.
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Effects of Amiloride, an Ion Channel Blocker, on Alamethicin Pore Formation in Negatively Charged, Gold-Supported, Phospholipid Bilayers: A Molecular View
2019Co-Authors: Fatemeh Abbasi, Jay J. Leitch, Julia Alvarez-malmagro, Jacek LipkowskiAbstract:The effects of amiloride on the structure and conductivity of Alamethicin ion pore formation within negatively charged, gold-supported, 1,2-dimyristoyl-sn-glycero-3-phosphocholine/Egg-PG membranes were investigated with the help of electrochemical impedance spectroscopy (EIS), photon polarization modulation-infrared reflection spectroscopy (PM-IRRAS), and atomic force microscopy (AFM). The EIS results indicate that ion conductivity across negatively charged phospholipid bilayers containing Alamethicin decreases by an order of magnitude when amiloride is introduced to the system. Despite the reduction in ion conductivity, the PM-IRRAS data shows that amiloride does not inhibit ion channel formation by Alamethicin peptides. High-resolution AFM images revealed that amiloride enlarges and distorts the shape of Alamethicin ion pores when introduced to the system, indicating that it is inserting itself into the mouth of the Alamethicin pores. This effect is driven by electrostatic interactions between positively charged amiloride molecules and the negative charge on the membrane
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pore forming properties of Alamethicin in negatively charged floating bilayer lipid membranes supported on gold electrodes
Langmuir, 2018Co-Authors: Fatemeh Abbasi, Jay J. Leitch, Julia Alvarezmalmagro, Jacek LipkowskiAbstract:Electrochemical impedance spectroscopy (EIS), atomic force microscopy (AFM), and photon polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) were employed to investigate the formation of Alamethicin pores in negatively charged bilayers composed of a mixture of 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) and egg-PG floating at gold (111) electrode surfaces modified by self-assembled monolayers of 1-thio-β-d-glucose (β-Tg). The EIS data showed that the presence of Alamethicin decreases the membrane resistivity by about 1 order of magnitude. PM-IRRAS measurements provided information about the tilt angles of peptide helical axis with respect to the bilayer normal. The small tilt angles obtained for the peptide helical axis prove that the Alamethicin molecules were inserted into the DMPC/egg-PG membranes. The tilt angles decreased when negative potentials were applied, which correlates with the observed decrease in membrane resistivity, indicating that ion pore formation is assisted by the transmembrane potential. Molecular resolution AFM images provided visual evidence that Alamethicin molecules aggregate forming hexagonal porous 2D lattices with periodicities of 2.0 ± 0.2 nm. The pore formation by Alamethicin in the negatively charged membrane was compared with the interaction of this peptide with a bilayer formed by zwitterionic lipids. The comparison of these results showed that Alamethicin preferentially forms ion translocating pores in negatively charged phospholipid membranes.
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pore forming properties of Alamethicin in negatively charged floating bilayer lipid membranes supported on gold electrodes
Langmuir, 2018Co-Authors: Fatemeh Abbasi, Jay J. Leitch, Julia Alvarezmalmagro, Jacek LipkowskiAbstract:Electrochemical impedance spectroscopy (EIS), atomic force microscopy (AFM), and photon polarization modulation infrared reflection absorption spectroscopy (PM-IRRAS) were employed to investigate the formation of Alamethicin pores in negatively charged bilayers composed of a mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and egg-PG floating at gold (111) electrode surfaces modified by self-assembled monolayers of 1-thio-β-d-glucose (β-Tg). The EIS data showed that the presence of Alamethicin decreases the membrane resistivity by about 1 order of magnitude. PM-IRRAS measurements provided information about the tilt angles of peptide helical axis with respect to the bilayer normal. The small tilt angles obtained for the peptide helical axis prove that the Alamethicin molecules were inserted into the DMPC/egg-PG membranes. The tilt angles decreased when negative potentials were applied, which correlates with the observed decrease in membrane resistivity, indicating that ion pore formation is as...
Claudio Toniolo - One of the best experts on this subject based on the ideXlab platform.
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Alamethicin topology in phospholipid membranes by oriented solid state nmr and epr spectroscopies a comparison
Journal of Physical Chemistry B, 2009Co-Authors: Evgeniy S Salnikov, Claudio Toniolo, Fernando Formaggio, Marta De Zotti, J Raap, Joe D J Oʼneil, S A Dzuba, Burkhard BechingerAbstract:Alamethicin, a hydrophobic peptide that is considered a paradigm for membrane channel formation, was uniformly labeled with 15N, reconstituted into oriented phosphatidylcholine bilayers at concentrations of 1 or 5 mol %, and investigated by solid-state NMR spectroscopy as a function of temperature. Whereas the peptide adopts a transmembrane alignment in POPC bilayers at all temperatures investigated, it switches from a transmembrane to an in-plane orientation in DPPC membranes when passing the phase transition temperature. This behavior can be explained by an increase in membrane hydrophobic thickness and the resulting hydrophobic mismatch condition. Having established the membrane topology of Alamethicin at temperatures above and below the phase transition, ESEEM EPR was used to investigate the water accessibility of Alamethicin synthetic analogues carrying the electron spin label TOAC residue at one of positions 1, 8, or 16. Whereas in the transmembrane alignment the labels at positions 8 and 16 are scr...
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self aggregation of spin labeled Alamethicin in epc vesicles studied by pulsed electron electron double resonance
Journal of the American Chemical Society, 2007Co-Authors: Alexander D Milov, Claudio Toniolo, Fernando Formaggio, Rimma I Samoilova, Yuri D Tsvetkov, J RaapAbstract:The pulsed electron−electron double resonance technique was used to study the dipole−dipole interactions between 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid spin labels located at position 16 of an Alamethicin analogue in ePC vesicles that were frozen to 77 K. We show that under these conditions the Alamethicin molecules tend to form aggregates over the range of peptide concentrations 3.5 × 10-3 to 1 × 10-2 M. The number of molecules in the aggregate is found to be 4.2 ± 0.2. A spin-label distance distribution function is also obtained with a maximum at a distance of 2.3 nm and a half-height width of 1.3 nm. We envisage that these data will permit us to generate a molecular model of cellular ion channels
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pore forming properties of Alamethicin f50 5 inserted in a biological membrane
Chemistry & Biodiversity, 2007Co-Authors: Natascia Vedovato, Claudio Toniolo, Chiara Baldini, Giorgio RispoliAbstract:The pore-forming properties of native and synthetic Alamethicins were investigated in photoreceptor rod outer segments (OS) isolated from frog retina, and recorded in whole-cell configuration. The peptaibols were applied (and removed) to (from) the OS within less than 50 ms by means of a computer-controlled micro-perfusion system. Once blocked with light, the main OS endogenous conductance, the OS membrane resistance was >1 GOmega, allowing low-noise and high-resolution recordings. Currents of ca. 700 pA were recorded in symmetric K(+) (100 mM) and Ca(2+) (1 mM), upon applying 1 microM of Alamethicin F50/5 or its [L-Glu(OMe)(7,18,19)] analogue to the OS membrane (clamped at -20 mV). In the latter peptide, the Gln residues at positions 7, 18, and 19 were substituted with side-chain esterified Glu residues. For both peptides, the current activated exponentially, with a delay from peptide application, and exponentially returned to zero without any delay, upon removing the peptide from the external solution. The delay as well as the activation (tau(a)) and deactivation (tau(d)) time constants of the current produced by the modified Alamethicin were much slower, and the current noise was much larger, with respect to the corresponding values for Alamethicin F50/5. Therefore, the above three Gln residues are not a key factor for pore formation, but the [L-Glu(OMe)(7,18,19)] analogue produces larger pores with a lower probability of formation.
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lipid chain length dependence for incorporation of Alamethicin in membranes electron paramagnetic resonance studies on toac spin labeled analogs
Biophysical Journal, 2007Co-Authors: Micha Jost, Cristina Peggion, Claudio TonioloAbstract:Alamethicin is a 19-residue hydrophobic peptide, which is extended by a C-terminal phenylalaninol but lacks residues that might anchor the ends of the peptide at the lipid-water interface. Voltage-dependent ion channels formed by Alamethicin depend strongly in their characteristics on chain length of the host lipid membranes. EPR spectroscopy is used to investigate the dependence on lipid chain length of the incorporation of spin-labeled Alamethicin in phosphatidylcholine bilayer membranes. The spin-label amino acid TOAC is substituted at residue positions n = 1, 8, or 16 in the sequence of Alamethicin F50/5 [TOACn, Glu(OMe)7,18,19]. Polarity-dependent isotropic hyperfine couplings of the three TOAC derivatives indicate that Alamethicin assumes approximately the same location, relative to the membrane midplane, in fluid diCNPtdCho bilayers with chain lengths ranging from N = 10–18. Residue TOAC8 is situated closest to the bilayer midplane, whereas TOAC16 is located farther from the midplane in the hydrophobic core of the opposing lipid leaflet, and TOAC1 remains in the lipid polar headgroup region. Orientational order parameters indicate that the tilt of Alamethicin relative to the membrane normal is relatively small, even at high temperatures in the fluid phase, and increases rather slowly with decreasing chain length (from 13° to 23° for N = 18 and 10, respectively, at 75°C). This is insufficient for Alamethicin to achieve hydrophobic matching. Alamethicin differs in its mode of incorporation from other helical peptides for which transmembrane orientation has been determined as a function of lipid chain length.
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supramolecular structure of self assembling Alamethicin analog studied by esr and peldor
Chemistry & Biodiversity, 2007Co-Authors: Alexander D Milov, Micha Jost, Cristina Peggion, Claudio Toniolo, Fernando Formaggio, Yuri D Tsvetkov, Janwillem Handgraaf, Marina I Samoilova, Marco Crisma, J RaapAbstract:Three analogs of Alamethicin F50/5, labelled with the TOAC (='2,2,6,6-tetramethylpiperidin-1-oxyl-4-amino-4-carboxylic acid') spin label at positions 1 (Alm1), 8 (Alm8), and 16 (Alm16), resp., were studied by Electron-Spin-Resonance (ESR) and Pulsed Electron-Electron Double-Resonance (PELDOR) techniques in solvents of different polarity to investigate the self-assembly of amphipathic helical peptides in membrane-mimicking environments. In polar solvents, Alamethicin forms homogeneous solutions. In the weakly polar chloroform/toluene 1 : 1 mixture, however, this peptide forms aggregates that are detectable at 293 K by ESR in liquid solution, as well as by PELDOR in frozen, glassy solution at 77 K. In liquid solution, free Alamethicin molecules and their aggregates show rotational-mobility correlation times tau(r) of 0.87 and 5.9 ns, resp. Based on these values and analysis of dipole-dipole interactions of the TOAC labels in the aggregates, as determined by PELDOR, the average number N of Alamethicin molecules in the aggregates is estimated to be less than nine. A distance-distribution function between spin labels in the supramolecular aggregate was obtained. This function exhibits two maxima: a broad one at a distance of 3.0 nm, and a wide one at a distance of ca. 7 nm. A molecular-dynamics (MD)-based model of the aggregate, consisting of two parallel tetramers, each composed of four molecules arranged in a 'head-to-tail' fashion, is proposed, accounting for the observed distances and their distribution.
Niels Chr Nielsen - One of the best experts on this subject based on the ideXlab platform.
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mechanisms of peptide induced pore formation in lipid bilayers investigated by oriented 31p solid state nmr spectroscopy
PLOS ONE, 2012Co-Authors: Kresten Bertelsen, Niels Chr Nielsen, Jerzy Dorosz, Sara K Hansen, Thomas VosegaardAbstract:There is a considerable interest in understanding the function of antimicrobial peptides (AMPs), but the details of their mode of action is not fully understood. This motivates extensive efforts in determining structural and mechanistic parameters for AMP’s interaction with lipid membranes. In this study we show that oriented-sample 31P solid-state NMR spectroscopy can be used to probe the membrane perturbations and -disruption by AMPs. For two AMPs, Alamethicin and novicidin, we observe that the majority of the lipids remain in a planar bilayer conformation but that a number of lipids are involved in the peptide anchoring. These lipids display reduced dynamics. Our study supports previous studies showing that Alamethicin adopts a transmembrane arrangement without significant disturbance of the surrounding lipids, while novicidin forms toroidal pores at high concentrations leading to more extensive membrane disturbance.
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cyclodextrin scaffolded Alamethicin with remarkably efficient membrane permeabilizing properties and membrane current conductance
Journal of Physical Chemistry B, 2012Co-Authors: Claudia U Hjorringgaard, Brian S Vad, Daniel E Otzen, Niels Chr Nielsen, Vladimir V Matchkov, Soren B Nielsen, Thomas Vosegaard, Troels SkrydstrupAbstract:Bacterial resistance to classical antibiotics is a serious medical problem, which continues to grow. Small antimicrobial peptides represent a potential solution and are increasingly being developed as novel therapeutic agents. Many of these peptides owe their antibacterial activity to the formation of trans-membrane ion-channels resulting in cell lysis. However, to further develop the field of peptide antibiotics, a thorough understanding of their mechanism of action is needed. Alamethicin belongs to a class of peptides called peptaibols and represents one of these antimicrobial peptides. To examine the dynamics of assembly and to facilitate a thorough structural evaluation of the Alamethicin ion-channels, we have applied click chemistry for the synthesis of templated Alamethicin multimers covalently attached to cyclodextrin-scaffolds. Using oriented circular dichroism, calcein release assays, and single-channel current measurements, the α-helices of the templated multimers were demonstrated to insert int...
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peptide aggregation and pore formation in a lipid bilayer a combined coarse grained and all atom molecular dynamics study
Biophysical Journal, 2008Co-Authors: Niels Chr Nielsen, Thomas Vosegaard, Lea Thogersen, Birgit Schiott, Emad TajkhorshidAbstract:We present a simulation study where different resolutions, namely coarse-grained (CG) and all-atom (AA) molecular dynamics simulations, are used sequentially to combine the long timescale reachable by CG simulations with the high resolution of AA simulations, to describe the complete processes of peptide aggregation and pore formation by Alamethicin peptides in a hydrated lipid bilayer. In the 1-μs CG simulations the peptides spontaneously aggregate in the lipid bilayer and exhibit occasional transitions between the membrane-spanning and the surface-bound configurations. One of the CG systems at t = 1 μs is reverted to an AA representation and subjected to AA simulation for 50 ns, during which water molecules penetrate the lipid bilayer through interactions with the peptide aggregates, and the membrane starts leaking water. During the AA simulation significant deviations from the α-helical structure of the peptides are observed, however, the size and arrangement of the clusters are not affected within the studied time frame. Solid-state NMR experiments designed to match closely the setup used in the molecular dynamics simulations provide strong support for our finding that Alamethicin peptides adopt a diverse set of configurations in a lipid bilayer, which is in sharp contrast to the prevailing view of Alamethicin oligomers formed by perfectly aligned helical Alamethicin peptides in a lipid bilayer.
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conformation of Alamethicin in oriented phospholipid bilayers determined by 15n solid state nuclear magnetic resonance
Biophysical Journal, 2001Co-Authors: Mads Bak, Robert P Bywater, Morten Hohwy, Jens K Thomsen, Kim Adelhorst, Hans J Jakobsen, Ole W Sorensen, Niels Chr NielsenAbstract:Abstract The conformation of the 20-residue antibiotic ionophore Alamethicin in macroscopically oriented phospholipid bilayers has been studied using 15 N solid-state nuclear magnetic resonance (NMR) spectroscopy in combination with molecular modeling and molecular dynamics simulations. Differently 15 N-labeled variants of Alamethicin and an analog with three of the α -amino-isobutyric acid residues replaced by alanines have been investigated to establish experimental structural constraints and determine the orientation of Alamethicin in hydrated phospholipid (dimyristoylphosphatidylcholine) bilayers and to investigate the potential for a major kink in the region of the central Pro 14 residue. From the anisotropic 15 N chemical shifts and 1 H– 15 N dipolar couplings determined for Alamethicin with 15 N-labeling on the Ala 6 , Val 9 , and Val 15 residues and incorporated into phospholipid bilayer with a peptide:lipid molar ratio of 1:8, we deduce that Alamethicin has a largely linear α -helical structure spanning the membrane with the molecular axis tilted by 10–20° relative to the bilayer normal. In particular, we find compatibility with a straight α -helix tilted by 17° and a slightly kinked molecular dynamics structure tilted by 11° relative to the bilayer normal. In contrast, the structural constraints derived by solid-state NMR appear not to be compatible with any of several model structures crossing the membrane with vanishing tilt angle or the earlier reported x-ray diffraction structure (Fox and Richards, Nature. 300:325–330, 1982). The solid-state NMR-compatible structures may support the formation of a left-handed and parallel multimeric ion channel.
Huey W. Huang - One of the best experts on this subject based on the ideXlab platform.
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Structure of the Alamethicin Pore Reconstructed by X-Ray Diffraction Analysis
Biophysical Journal, 2008Co-Authors: Shuo Qian, Wangchen Wang, Lin Yang, Huey W. HuangAbstract:We reconstructed the electron density profile of the Alamethicin-induced transmembrane pore by x-ray diffraction. We prepared fully hydrated multiple bilayers of Alamethicin-lipid mixtures in a condition where pores were present, as established previously by neutron in-plane scattering in correlation with oriented circular dichroism. At dehydrated conditions, the interbilayer distance shortened and the interactions between bilayers caused the membrane pores to become long-ranged correlated and form a periodically ordered lattice of rhombohedral symmetry. To resolve the phase problem of diffraction, we used a brominated lipid and performed multiwavelength anomalous diffraction at the bromine K edge. The result unambiguously shows that the Alamethicin pore is of the barrel-stave type consisting of eight Alamethicin helices. This pore structure corresponds to the stable pores detected by neutron in-plane scattering in fully hydrated fluid bilayers at high peptide/lipid ratios, which are the conditions at which Alamethicin was tested for its antibacterial activity.
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barrel stave model or toroidal model a case study on melittin pores
Biophysical Journal, 2001Co-Authors: Lin Yang, Thad A Harroun, Thomas M Weiss, Lai Ding, Huey W. HuangAbstract:Transmembrane pores induced by amphiphilic peptides, including melittin, are often modeled with the barrel-stave model after the Alamethicin pore. We examine this assumption on melittin by using two methods, oriented circular dichroism (OCD) for detecting the orientation of melittin helix and neutron scattering for detecting transmembrane pores. OCD spectra of melittin were systematically measured. Melittin can orient either perpendicularly or parallel to a lipid bilayer, depending on the physical condition and the composition of the bilayer. Transmembrane pores were detected when the helices oriented perpendicularly to the plane of the bilayers, not when the helices oriented parallel to the bilayers. The evidence that led to the barrel-stave model for Alamethicin and that to the toroidal model for magainin were reviewed. The properties of melittin pores are closely similar to that of magainin but unlike that of Alamethicin. We conclude that, among naturally produced peptides that we have investigated, only Alamethicin conforms to the barrel-stave model. Other peptides, including magainins, melittin and protegrins, all appear to induce transmembrane pores that conform to the toroidal model in which the lipid monolayer bends continuously through the pore so that the water core is lined by both the peptides and the lipid headgroups.
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Mechanism of Alamethicin insertion into lipid bilayers
Biophysical Journal, 1996Co-Authors: Steven J. Ludtke, William T Heller, Huey W. HuangAbstract:Alamethicin adsorbs on the membrane surface at low peptide concentrations. However, above a critical peptide-to-lipid ratio (P/L), a fraction of the peptide molecules insert in the membrane. This critical ratio is lipid dependent. For diphytanoyl phosphatidylcholine it is about 1/40. At even higher concentrations P/L > or = 1/15, all of the Alamethicin inserts into the membrane and forms well-defined pores as detected by neutron in-plane scattering. A previous x-ray diffraction measurement showed that Alamethicin adsorbed on the surface has the effect of thinning the bilayer in proportion to the peptide concentration. A theoretical study showed that the energy cost of membrane thinning can indeed lead to peptide insertion. This paper extends the previous studies to the high-concentration region P/L > 1/40. X-ray diffraction shows that the bilayer thickness increases with the peptide concentration for P/L > 1/23 as the insertion approaches 100%. The thickness change with the percentage of insertion is consistent with the assumption that the hydrocarbon region of the bilayer matches the hydrophobic region of the inserted peptide. The elastic energy of a lipid bilayer including both adsorption and insertion of peptide is discussed. The Gibbs free energy is calculated as a function of P/L and the percentage of insertion phi in a simplified one-dimensional model. The model exhibits an insertion phase transition in qualitative agreement with the data. We conclude that the membrane deformation energy is the major driving force for the Alamethicin insertion transition.
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Neutron scattering in the plane of membranes: structure of Alamethicin pores
Biophysical Journal, 1996Co-Authors: Steven J. Ludtke, David L. Worcester, Huey W. HuangAbstract:A technique of neutron in-plane scattering for studying the structures of peptide pores in membranes is described. Alamethicin in the inserted state was prepared and undeuterated and deuterated dilauroyl phosphatidylcholine (DLPC) hydrated with D2O or H2O. Neutron in-plane scattering showed a strong dependence on deuteration, clearly indicating that water is a part of the high-order structure of inserted Alamethicin. The data are consistent with the simple barrel-stave model originally proposed by Baumann and Mueller. The theoretical curves computed with this model at four different deuteration conditions agree with the data in all cases. Both the diameter of the water pore and the effective outside diameter of the channel are determined accurately. Alamethicin forms pores in a narrow range of size. In a given sample condition, > 70% of the peptide forms pores of n and n +/- 1 monomers. The pore size varies with hydration and with lipid. In DLPC, the pores are made of n = 8–9 monomers, with a water pore approximately 18 A in diameter and with an effective outside diameter of approximately 40 A. In diphytanoyl phosphatidylcholine, the pores are made of n approximately 11 monomers, with a water pore approximately 26 A in diameter, with an effective outside diameter of approximately 50 A.
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antimicrobial peptide pores in membranes detected by neutron in plane scattering
Biochemistry, 1995Co-Authors: Ke He, Huey W. Huang, S J Ludtke, David L. WorcesterAbstract:Antimicrobial peptides isolated from the host defense systems of animals have been shown to exert their activity directly on the lipid bilayer of cell membranes, but the antimicrobial mechanisms are not clear, due chiefly to the difficulty of discerning the high-order structures formed by these peptides in membranes. Previously we have shown that these peptides insert into the membrane when their concentrations exceed a lipid-dependent critical value. With neutron in-plane scattering we now show that inserted Alamethicin creates aqueous pores 218 A in diameter. The density of pores is consistent with the assumption that all of the Alamethicin is involved in pore formation. Pores were not detected below the critical concentration. Thus concentration-dependent pore formation appears to be the molecular mechanism of antimicrobial action.