Influence of Lipid Saturation,Hydrophobic Length and Cholesterol on Double-Arginine-Containing Helical Peptides in Bilayer Membranes

Membrane proteins are essential for many cell processes yet are more difficult to investigate than soluble proteins. Charged residues often contribute significantly to membrane protein function. Model peptides such as GWALP23 (acetyl-GGALW⁵LAL⁸LALALAL¹⁶ALW¹⁹LAGA-amide) can be used to characterize the influence of specific residues on transmembrane protein domains. We have substituted R8 and R16 in GWALP23 in place of L8 and L16, equidistant from the peptide center, and incorporated specific ²H-labeled alanine residues within the central sequence for detection by solid-state ²H NMR spectroscopy. The resulting pattern of [²H]Ala quadrupolar splitting (Δν_q) magnitudes indicates the core helix for R^8,16GWALP23 is significantly tilted to give a similar transmembrane orientation in thinner bilayers with either saturated C12:0 or C14:0 acyl chains (1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)) or unsaturated C16:1 Δ9 cis acyl chains. In bilayers of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC; C18:1 Δ9 cis) multiple orientations are indicated, whereas in longer, unsaturated 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEiPC; C20:1 Δ11 cis) bilayers, the R^8,16GWALP23 helix adopts primarily a surface orientation. The inclusion of 10–20 mol % cholesterol in DOPC bilayers drives more of the R^8,16GWALP23 helix population to the membrane surface, thereby allowing both charged arginines access to the interfacial lipid head groups. The results suggest that hydrophobic thickness and cholesterol content are more important than lipid saturation for the arginine peptide dynamics and helix orientation in lipid membranes.     

Temperature-Dependent Re-alignment of the Short Multifunctional Peptide BP100 in Membranes Revealed by Solid-State NMR Spectroscopy and Molecular Dynamics Simulations

BP100 is a cationic undecamer peptide with antimicrobial and cell-penetrating activities. The orientation of this amphiphilic α-helix in lipid bilayers was examined under numerous conditions using solid-state ¹⁹F, ¹⁵N and ²H NMR. At high temperatures in saturated phosphatidylcholine lipids, BP100 lies flat on the membrane surface, as expected. Upon lowering the temperature towards the lipid phase transition, the helix is found to flip into an upright transmembrane orientation. In thin bilayers, this inserted state was stable at low peptide concentration, but thicker membranes required higher peptide concentrations. In the presence of lysolipids, the inserted state prevailed even at high temperature. Molecular dynamics simulations suggest that BP100 monomer insertion can be stabilized by snorkeling lysine side chains. These results demonstrate that even a very short helix like BP100 can span (and thereby penetrate through) a cellular membrane under suitable conditions.     

Interactions of KLA Amphipathic Model Peptides with Lipid Monolayers

Interactions of the cationic amphipathic peptide KLALKLALKALKAALKLA‐NH₂ (KLAL) and its double D ‐amino acid replacement analogues l¹¹k¹²‐KLAL and k⁹a¹⁰‐KLAL with lipid monolayers of anionic POPG, zwitterionic POPC and mixtures thereof at the air/water interface were investigated by infrared reflection– absorption spectroscopy (IRRAS). At high surface pressure (>30 mN m^?1) all peptides incorporated into lipid monolayers containing at least 25 % anionic POPG, and adopted an α‐helical conformation. Creation of free surface by expansion of the monolayers resulted in an additional adsorption of peptides from the subphase, but now in a β‐sheet conformation; this led to the coexistence of peptides in two distinctly different conformations within the lipid monolayer. The β‐sheets bound to the free surface could be squeezed out of the film by compressing the film to low surface areas, whereas the α‐helices remained bound to the lipids until the film collapsed. When bound to the lipid monolayer, the helical axis of the peptides is oriented almost parallel to the surface of the monolayer.     

Fabrication and optical characterization of poly(2,5‐di‐n‐butoxyphenylene) nanofibril arrays

Anodic aluminum oxide (AAO) membrane can be used as template for the synthesized nanostructures. In this article, we have prepared the AAO membrane by using electrooxidation of aluminum substrate in phosphoric acid, and fabricated poly(2,5‐di‐n‐butoxyphenylene) (BuO–PPP) nanofibril arrays by oxidative coupling polymerization of 1,4‐di‐n‐butoxybenzene (DBB) within the pores of the AAO template membrane. The detailed molecular structure of the polymer nanofibrils was characterized by using infrared and ¹H nuclear magnetic resonance spectra, and estimated to consist of almost equal fractions of 1,4‐ and 1,3‐ linkages. We have used transmission electron microscopy, scanning electron microscopy, and atom force microscopy to confirm the morphologies and images of the AAO template membrane and the fabricated nanometer scale of BuO–PPP nanofibril arrays. The experimental results demonstrated that the pores of the AAO membrane were regular and uniform, and parallel each other, and the BuO–PPP chains in the narrowest template‐synthesized nanofibrils were oriented parallel to the porous axes of the AAO membrane and perpendicular to the surface of the aluminum substrate. The polymer chain orientation was partially responsible for the enhanced conductivity. The ultraviolet absorption spectrum of the BuO–PPP nanofibril arrays shown that the polymer contains a better extended π‐conjugation system along poly‐(p‐phenylene) backbone, which resulted in longer wavelength shift of the absorption band, the absorption maxima were located at 258 nm (E₁ absorption band) and 332 nm (E₂ absorption band), respectively. Photoluminescence spectrum of the BuO–PPP nanofibril arrays exhibited a blue emission. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 91: 425–430, 2004     

Probing and Manipulating the Lateral Pressure Profile in Lipid Bilayers Using Membrane-Active Peptides—A Solid-State 19F NMR Study

The lateral pressure profile constitutes an important physical property of lipid bilayers, influencing the binding, insertion, and function of membrane-active peptides, such as antimicrobial peptides. In this study, we demonstrate that the lateral pressure profile can be manipulated using the peptides residing in different regions of the bilayer. A ¹⁹F-labeled analogue of the amphiphilic peptide PGLa was used to probe the lateral pressure at different depths in the membrane. To evaluate the lateral pressure profile, we measured the orientation of this helical peptide with respect to the membrane using solid-state ¹⁹F-NMR, which is indicative of its degree of insertion into the bilayer. Using this experimental approach, we observed that the depth of insertion of the probe peptide changed in the presence of additional peptides and, furthermore, correlated with their location in the membrane. In this way, we obtained a tool to manipulate, as well as to probe, the lateral pressure profile in membranes.     

Simple Systematic Synthesis of Periodic Mesoporous Organosilica Nanoparticles with Adjustable Aspect Ratios

One-dimensional periodic mesoporous organosilica (PMO) nanoparticles with tunable aspect ratios are obtained from a chain-type molecular precursor octaethoxy-1,3,5-trisilapentane. The aspect ratio can be tuned from 2:1 to >20:1 simply by variation in the precursor concentration in acidic aqueous solutions containing constant amounts of triblock copolymer Pluronic P123. The mesochannels are highly ordered and are oriented parallel to the longitudinal axis of the PMO particles. No significant Si–C bond cleavage occurs during the synthesis according to²⁹Si MAS NMR. The materials exhibit surface areas between 181 and 936 m² g⁻¹.     

Solution Structure of the Leader Sequence of the Patellamide Precursor Peptide,PatE1–34

The solution structure of the leader sequence of the patellamide precursor peptide was analysed by using CD and determined with NOE‐restrained molecular dynamics calculations. This leader sequence is highly conserved in the precursor peptides of some other cyanobactins harbouring heterocycles, and is assumed to play a role in targeting the precursor peptide to the post‐translational machinery. The sequence was observed to form an α‐helix spanning residues 13–28 with a hydrophobic surface on one side of the helix. This hydrophobic surface is proposed to be the site of the initial binding with modifying enzymes.     

Involvement of Acidic Amino Acid Residues in Zn2+ Binding to Respiratory Complex I

Proton transfer across membranes and membrane proteins is a central process in biological systems. Zn²⁺ ions are capable of binding to acidic residues, often found within such specific pathways, thereby leading to a blockage. Here we probed Zn²⁺inhibition of the proton‐pumping NADH:ubiquinone oxidoreductase from Escherichia coli by means of electrochemically induced FTIR difference spectroscopy. Numerous conformational changes were identified including those that arise from the reorganization of the membrane arm upon electron transfer in the peripheral arm of the protein. Signals at very high wavenumbers (1781 and 1756 cm^?1) point to the perturbation of acidic residues in a highly hydrophobic environment upon Zn²⁺ binding. In variant D563N^L, which lacks part of the proton pumping activity (residue located on the horizontal amphipathic helix), the spectral signature of Zn²⁺ binding is changed. Our data support a role for this residue in proton translocation.     

Preparation and characterization of highly oriented poly(γ‐benzyl L‐glutamate) networks and gels with long channels with micrometer‐scale diameters

Poly(γ‐benzyl L ‐glutamate) (PBLG) gels with highly oriented α‐helix chains were prepared by the crosslinking of PBLG chains through changes in the concentration of ethylenediamine, used as a crosslinker, in 1,4‐dioxane in the presence of the strong magnetic field of an NMR magnet with a strength of 10.5 T. The experimental results showed that in one of these gels, long channels with an average diameter of about 100 μm were formed by phase separation between crosslinked PBLG and the solvent. Furthermore, three‐dimensional ¹H‐NMR imaging patterns showed that the long channels were aligned in the direction parallel to the α‐helix axis. The PBLG gel was swollen in the direction perpendicular to the α‐helix axis, but it was not swollen in the direction parallel to the α‐helix axis. The X‐ray diffraction patterns of the gel showed that the interchain distance between the two nearest neighboring PBLG chains changed from 13.4 to 18.1 Å with a change in the swelling degree. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: 1053–1060, 2004     

Juxta‐terminal Helix Unwinding as a Stabilizing Factor to Modulate the Dynamics of Transmembrane Helices

Transmembrane helices of integral membrane proteins often are flanked by interfacial aromatic residues that can serve as anchors to aid the stabilization of a tilted transmembrane orientation. Yet, physical factors that govern the orientation or dynamic averaging of individual transmembrane helices are not well understood and have not been adequately explained. Using solid‐state ²H NMR spectroscopy to examine lipid bilayer‐incorporated model peptides of the GWALP23 (acetyl‐GGALW(LA)₆LWLAGA‐amide) family, we observed substantial unwinding at the terminals of several tilted helices spanning the membranes of DLPC, DMPC, or DOPC lipid bilayers. The fraying of helix ends might be vital for defining the dynamics and orientations of transmembrane helices in lipid bilayer membranes.     

Metal Binding Properties of Fluorescent Analogues of Trichogin GA IV: A Conformational Study by Time‐Resolved Spectroscopy and Molecular Mechanics Investigations

The metal ion binding properties of two fluorescent analogues of trichogin GA IV, which is a natural undecapeptide showing significant antimicrobial activity, were studied by circular dichroism, time‐resolved optical spectroscopy, and molecular mechanics calculations. Binding of Ca^II and Gd^III to the peptides investigated was shown to promote a structural transition from highly helical conformations to folded structures characterized by formation of a loop that embedded the metal ion. Time‐resolved spectroscopy revealed that peptide dynamics is also remarkably affected by ion binding: peptide‐backbone motions slowed down to the microsecond time scale. Finally, molecular mechanics calculations emphasized the role of the central Gly5‐Gly6 motif, which allowed for the twisting of the peptide segment that gave rise to the formation of the binding cavity.     

Selective Antifungal Activity and Fungal Biofilm Inhibition of Tryptophan Center Symmetrical Short Peptide

Candida albicans, an opportunistic fungus, causes dental caries and contributes to mucosal bacterial dysbiosis leading to a second infection. Furthermore, C.albicans forms biofilms that are resistant to medicinal treatment. To make matters worse, antifungal resistance has spread (albeit slowly) in this species. Thus, it has been imperative to develop novel, antifungal drug compounds. Herein, a peptide was engineered with the sequence of RRFSFWFSFRR-NH2; this was named P19. This novel peptide has been observed to exert disruptive effects on fungal cell membrane physiology. Our results showed that P19 displayed high binding affinity to lipopolysaccharides (LPS), lipoteichoic acids (LTA) and the plasma membrane phosphatidylinositol (PI), phosphatidylserine (PS), cardiolipin, and phosphatidylglycerol (PG), further indicating that the molecular mechanism of P19 was not associated with the receptor recognition, but rather related to competitive interaction with the plasma membrane. In addition, compared with fluconazole and amphotericin B, P19 has been shown to have a lower potential for resistance selection than established antifungal agents.     

De novo design of a peptide which partitions between water and phospholipid bilayers as a monomeric alpha-helix

To dissect the determinants of protein insertion into membranes, we designed a model peptide which partitions between water and phospholipid bilayers as an alpha-helical monomer. We used a simplex method to optimize the 'a, d hydrophobicity' and 'e, g charge' of a series of five peptides, where 'abcdefg' correspond to the positions in two turns of an alpha-helix. Circular dichroism and analytical ultra- centrifugation experiments showed that the final peptide (helix5) is monomeric and has an alpha-helix content of approximately 89% at 0 degrees C in aqueous solution. In the presence of large unilamellar vesicles (LUVs), helix5 partitions between the aqueous and membranous phases with a partition constant well suited for measurements by electron paramagnetic resonance (EPR) spectroscopy. EPR power saturation experiments with a cysteine-scanning strategy showed that the alpha-helicity of helix5 is conserved upon binding to LUVs and that the alpha-helix binds parallel to the membrane surface with the central axis approximately 5 A below the lipid phosphate groups. Helix5 should be a useful model peptide for studies aimed at dissecting the determinants of the membrane binding of alpha-helices. The simplex- based strategy may be useful in the rational design of proteins when desired structural or partitioning properties cannot be selected or screened from libraries.      

A Temperature-Driven,Reversible, Helical-Handedness Inversion in Peptaibol Analogues Tuned by the C-Terminal Capping Moiety

Trichogin is a natural peptide endowed with antimicrobial and antitumor activity. A member of the peptaibol family, trichogin possesses a C-terminal amino alcohol. In the past, this moiety was substituted for a methyl ester for synthetic purposes and it was observed that this apparently slight modification caused significant changes in the peptide bioactivity. With the aim of understanding the reasons behind such observations, a detailed spectroscopic study on a number of trichogin analogues has been performed. Herein, data obtained from synchrotron radiation circular dichroism, NMR spectroscopy, and fluorescence spectroscopy in organic solvents at cryogenic temperatures are compared with those independently acquired by means of EPR spectroscopy at 80 K. It is unambiguously revealed that the presence of a reversible, temperature-driven, screw-sense interconversion from a right- to left-handed helix is determined by the C-terminal capping moiety. Data demonstrate, for the first time, the key role of a C-terminal methyl ester in promoting peptide screw-sense inversion.     

Nafion-clay nanocomposite membranes: Morphology and properties

A series of Nafion-clay nanocomposite membranes were synthesized and characterized. To minimize any adverse effects on ionic conductivity the clay nanoparticles were H⁺ exchanged prior to mixing with Nafion. Well-dispersed, mechanically robust, free-standing nanocomposite membranes were prepared by casting from a water suspension at 180 °C under pressure. SAXS profiles reveal a preferential orientation of Nafion aggregates parallel to the membrane surface, or normal plane. This preferred orientation is induced by the platy nature of the clay nanoparticles, which tend to align parallel to the surface of the membrane. The nanocomposite membranes show dramatically reduced methanol permeability, while maintaining high levels of proton conductivity. The hybrid films are much stiffer and can withstand much higher temperatures compared to pure Nafion. The superior thermomechanical, electrochemical and barrier properties of the nanocomposite membranes are of significant interest for direct methanol fuel cell applications.     

Tethered bimolecular lipid membranes—A novel model membrane platform

This contribution summarizes some of our efforts in designing, synthesizing, assembling, and characterizing functional tethered bimolecular lipid membranes (tBLMs) as a novel platform for biophysical studies of and with artificial membranes or for sensor development employing, e.g., membrane integral receptor proteins. Chemical coupling schemes based on thiol groups for Au substrates or silanes used in the case of oxide surfaces allow for the covalent and, hence, chemically and mechanically robust attachment of anchor lipids to the solid support, stabilizing the proximal layer of a tethered membrane on the transducer surface. Surface plasmon optics, the quartz crystal microbalance, fluorescence- and IR spectroscopies, and electrochemical techniques are used to characterize the build-up of these complex supramolecular interfacial architectures. We demonstrate, in particular, that bilayers with a specific electrical resistance of better than 10 MΩ cm² can be achieved routinely with this approach.The functionalization of the lipid membranes by the incorporation of peptides is demonstrated for the carrier valinomycin which shows in our tBLMs the expected discrimination by four orders of magnitude between the translocation of K⁺- and Na⁺-ions across the hydrophobic barrier. For the synthetic channel-forming peptide M2 the high electrical resistance of the bilayer with the correspondingly low background current allows for the recording of even single channel current fluctuations.From the many membrane proteins that we reconstituted so far we describe results obtained with the redox-protein cytochrome c oxidase. Here, we also use a genetically modified mutant with a His-tag at either the C- or the N-terminus for the oriented attachment of the protein via the NTA/Ni²⁺ approach. With this strategy, we not only can control the density of the immobilized functional units, we introduce a completely new and alternative concept for the stabilization of lipid bilayers, i.e., the protein-tethered membrane.Our efforts in experimentally characterizing the resulting membrane functions and correlating the data with the structural details of the bilayer architectures are complemented by theoretical studies modeling the electrical and electrochemical response of functional tethered lipid bilayer membranes by extended SPICE simulations.     

Buoyancy effect on the ultrafiltration of PEG 10,000. Visualization by digital holographic interferometry

Digital holographic interferometry has been used to visualize the buoyancy effects on dead‐end ultrafiltration of PEG 10,000. Sets of experiments have been carried out with the membrane in different orientations (90^°, 180^°), using a feed concentration of PEG 10,000 ranging 5–12.5 kg/m³. These results were compared with those obtained in previous research with the cell placed in its natural position (0^°). The interferometric fringe patterns obtained in this research were very different from those obtained previously. Whereas at the 0^° position, an increasing number of interferometric fringes appeared parallel to the membrane surface at 90 and 180^° positions, the number of interference fringes was much lesser and, with the 180^° orientation, slightly curved protuberances appeared after a short period of time that, like big drops, fell downward from the membrane surface. The consequence is a decrease of the polarization layer and an enhancement of the membrane performance. © 2012 American Institute of Chemical Engineers AIChE J, 2012     

Designed Di‐Heme Binding Helical Transmembrane Protein

De novo designing of functional membrane proteins is fundamental in terms of understanding the structure, folding, and stability of membrane proteins. In this work, we report the design and characterization of a transmembrane protein, termed HETPRO (HEme‐binding Transmembrane PROtein), that binds two molecules of heme in a membrane and catalyzes oxidation/reduction reactions. The primary structure of HETPRO has been optimized in a guided fashion, from an antimicrobial peptide, for transmembrane orientation, defined 3D structure, and functions. HETPRO assembles into a tetrameric form, from an apo dimeric helical structure, in complex with cofactor in detergent micelles. The NMR structure of the apo HETPRO in micelles reveals an antiparallel helical dimer that inserts into the nonpolar core of detergent micelles. The well‐defined structure of HETPRO and its ability to bind to heme moieties could be utilized to develop a functional membrane protein mimic for electron transport and photosystems.     

The Dynamics of Lysozyme from Bacteriophage Lambda in Solution Probed by NMR and MD Simulations

¹⁵N NMR relaxation studies, analyses of NMR data to include chemical shifts, residual dipolar couplings (RDC), NOEs and H^N–H^α coupling constants, and molecular dynamics (MD) simulations have been used to characterise the behaviour of lysozyme from bacteriophage lambda (λ lysozyme) in solution. The lower and upper lip regions in λ lysozyme (residues 51–60 and 128–141, respectively) show reduced ¹H–¹⁵N order parameters indicating mobility on a picosecond timescale. In addition, residues in the lower and upper lips also show exchange contributions to T₂ indicative of slower timescale motions. The chemical shift, RDC, coupling constant and NOE data for λ lysozyme indicate that two fluctuating β‐strands (β3 and β4) are populated in the lower lip region while the N terminus of helix α6 (residues 136–139) forms dynamic helical turns in the upper lip region. This behaviour is confirmed by MD simulations that show hydrogen bonds, indicative of the β‐sheet and helical secondary structure in the lip regions, with populations of 40–60 %. Thus in solution λ lysozyme adopts a conformational ensemble that will contain both the open and closed forms observed in the crystal structures of the protein.     

Direct fluorination of the polyimide matrimid® 5218: The formation kinetics and physicochemical properties of the fluorinated layers

Fluorinated polymers have a set of unique properties, including improved chemical stability and thermal stability and good barrier and membrane parameters, which are mainly defined by their surface properties. This article presents systematic data on the direct fluorination of the polyimide Matrimid® 5218, a commercially available polymer suitable for the formation of gas‐separation hollow fibers. Changing the fluorination conditions (i.e., the fluorinated mixture composition, fluorine partial pressure, and treatment duration) allows the rate of formation of the surface‐fluorinated layer over the 0.1–10 μm range to be kept under control. The physicochemical properties of modified layers (i.e., the chemical composition, formation of radicals, refractive index, IR and UV spectra, density, and surface energy) are examined. The thickness of the fluorinated layer (δ_F) depends on the fluorination duration (t): δ_F ～ t^0.5. During fluorination, hydrogen atoms are replaced with fluorine, double bonds are saturated with fluorine, and at least one CN bond in the five‐member ring is disrupted. Fluorination results in a significant increase in the polymer density, transparency in the visible and ultraviolet regions of spectra, and a reduction of the refractive index. A high concentration of long‐living radicals (up to ～5 × 10¹⁹ radicals/cm³ of the fluorinated layer) is generated under fluorination. This can be used for subsequent grafting (e.g., with acrylonitrile). © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 92: 6–17, 2004