Fluorescent, synthetic amphiphilic heptapeptide anion transporters: evidence for self-assembly and membrane localization in liposomes

Synthetic anion transporters (SATs) of the general type (n-C18H37)2N-COCH2OCH2CO-(Gly)3-Pro-(Gly)3-O-n-C7H15, 1, are amphiphilic peptides that form anion-conducting pores in bilayer membranes. To better understand membrane insertion, assembly and aggregation dynamics, and membrane penetration, four novel fluorescent structures were prepared for use in both aqueous buffer and phospholipid bilayers. The fluorescent residues pyrene, indole, dansyl, and NBD were incorporated into 1 to give 2, 3, 4, and 5, respectively. Assembly of peptide amphiphiles in buffer was confirmed by monitoring changes in the pyrene monomer/excimer peaks observed for 2. Solvent-dependent fluorescence changes that were observed for indole (3) and dansyl (4) side-chained SATs in bilayers showed that these residues experienced an environment between epsilon=9 (CH2Cl2) and epsilon=24 (EtOH) in polarity. Fluorescence resonance energy transfer (FRET) between 2 and 3 demonstrated aggregation of SAT monomers within the bilayer. This self-assembly led to pore formation, which was detected as Cl(-) release from the liposomes. The results of acrylamide quenching of fluorescent SATs supported membrane insertion. Studies with NBD-labeled SAT 5 showed that peptide partition into the bilayer is relatively slow. Dithionite quenching of NBD-SATs suggests that the amphiphilic peptides are primarily in the bilayer's outer leaflet. Images obtained by using a fluorescence microscope revealed membrane localization of a fluorescent SAT. Taken together, this study helps define the insertion, membrane localization, and aggregation behavior of this family of synthetic anion transporters in liposomal bilayers.     

Carboxylate anion diminishes chloride transport through a synthetic, self-assembled transmembrane pore

Six amphiphilic heptapeptides with the structure (C18H37)2NCOCH2OCH2CO-(Gly)3-Pro-(Gly)n-(Glx)-(Gly)m-O(CH2)6CH3, in which Glx represents glutamic acid or its benzyl ester and n+m=2, have been studied. In addition, the glutamate residue in the GGGPGGE sequence was esterified by fluorescent 1-pyrenemethanol. These compounds insert into phospholipid bilayers and form anion-conducting pores. Hill plots based on carboxyfluorescein release indicate that the pores are at least dimeric. Studies that involved ion-selective electrode techniques showed that transport of chloride varied with the position of glutamate within the peptide chain and whether glutamic acid was present as the free acid or its benzyl ester. Chloride transport activity was significantly higher for the glutamate esters than for free carboxylates irrespective of the glutamate position. Activity was highest when the glutamate residue in approximately (Gly)3-Pro-(Xxx)3 approximately was closest to the C terminus of the peptide. A fluorescent pyrene residue was introduced to probe the aggregation state of the amphiphile. The selectivity of the pore for Cl(-) over K+ was maintained even when the carboxylate anion was present within it. Complexation of Cl(-) by the ionophoric peptides was confirmed by negative ion mass spectrometry. Planar bilayer voltage clamp experiments confirmed that pores with more than one conductance state may form in these dynamic, self-assembled pores.     

Ligand-gated synthetic ion channels

Supramolecular pi-stack architecture is fundamental in DNA chemistry but absent in biological and synthetic ion channels and pores. Here, a novel rigid-rod pi-stack architecture is introduced to create synthetic ion channels with characteristics that are at the forefront of rational design, that is, ligand gating by a conformational change of the functional supramolecule. Namely, the intercalation of electron-rich aromatics is designed to untwist inactive electron-poor helical pi-stacks without internal space into open barrel-stave ion channels. Conductance experiments in planar lipid bilayers corroborate results from spherical bilayers and molecular modeling: Highly cooperative and highly selective ligand gating produces small, long-lived, weakly anion selective, ohmic ion channels. Structural studies conducted under conditions relevant for function provide experimental support for helix-barrel transition as origin of ligand gating. Control experiments demonstrate that minor structural changes leading to internal decrowding suffice to cleanly annihilate chiral self-organization and function.     

The Solution Structure and Self‐Association Properties of the Cyclic Lipodepsipeptide Pseudodesmin A Support Its Pore‐Forming Potential

Pseudodesmin A is a cyclic lipodepsipeptide (CLP) of the viscosin group with a moderate in vitro biological activity. For several CLPs, including members of this group, this activity has been related to the ability to form ion pores in cellular membranes. As their size does not allow individual CLPs to span the membrane bilayer, individual monomers must somehow assemble into a larger structure. NMR spectroscopy has been used to demonstrate that in chloroform and other apolar organic solvents, pseudodesmin A monomers assemble into a supramolecular structure. These self‐assembled structures can become sufficiently large to span the membrane bilayer as demonstrated with translational diffusion NMR spectroscopic measurements. With the aim to obtain more insight into the structural nature of this assembly, the solution conformation of pseudodesmin A was first determined by using ROESY (rOe) restraints measured in acetonitrile, in which no self‐association occurs. The structure, which is found to be mostly similar to the previously described crystal structure, is shown to be retained within the supramolecular complex. Intermolecular rOe contacts obtained in chloroform together with chemical shift perturbation data provides structural insight into the organization of the self‐associated complex. Based upon this analysis, a model for the organization of pseudodesmin A monomers in the supramolecular assembly is proposed, which is in agreement with the formation of bilayer spanning hydrophilic pores and provides the basis for a structure–function relationship for this type of CLPs. Finally, it is demonstrated that the differences previously reported between the crystal and solution conformation of the white line inducing principle (WLIP), a close analogue of pseudodesmin A, are the result of the use of dimethyl sulfoxide as solvent, whose strong hydrogen‐bonding capacity induces conformational exchange.     

Aggregation behavior and dynamics of synthetic amphiphiles that self-assemble to anion transporters

The amphiphilic heptapeptides-referred to as synthetic anion transporters (SATs)-mediate chloride transport in planar lipid bilayer membranes, synthetic liposomes, and mammalian cells. The SATs described have the general formula R1(2)NCOCH2OCH2CO-(Gly)3-Pro-(Gly)3-OR2. Substitution at R1 and R2 with various aliphatic or aromatic groups alters the ability of SATs to transport chloride through a phospholipid bilayer membrane. Despite extensive structure-activity relationship studies concerning Cl(-)-mediated transport by SATs, relatively little was known about the mechanism of insertion and pore-formation in the membrane. In the current study, the mechanistic behavior of SATs was investigated in aqueous solution and at the air-water interface. In the latter case, Langmuir trough studies and Brewster angle microscopy (BAM) revealed the extent of monolayer stability and organization for SATs. Dynamic light scattering and transmission electron microscopy (TEM) confirmed these results and defined the aggregation behavior of SATs in solution. SAT derivatives that showed low chloride transport activity organized into stable monolayers at the air-water interface, while more active SATs formed less stable monolayers. The relationship between intermolecular organization of SATs and pore-formation in the membrane is discussed along with its implications for chloride transport.     

Palladium(II)-mediated assembly of biotinylated ion channels

Simple synthetic methodology has been used to create biotinylated pyridyl cholate lipids that can undergo multiple self-assembly events when inserted into phospholipid vesicles; Pd(II) links cholates into transmembrane lipids, while avidin laterally clusters these complexes together and concomitantly assembles the vesicles into aggregates. The transmembrane assembly of cholates by Pd(II) "opened" the ion channels, whereas avidin addition produced vesicle aggregates, giving a system that mimicked both transmembrane transport and cellular adhesion. Complexation of these Pd(II)-linked cholates by avidin gave a measurable decrease in ion flow, suggesting some channels became blocked or were prevented from adopting the optimum geometry for ion conduction. This reflects the importance of spatially appropriate preorganisation when generating active supramolecular assemblies.     

Reversible Immobilization of Peptides: Surface Modification and In Situ Detection by Attenuated Total Reflection FTIR Spectroscopy

A generic method is described for the reversible immobilization of polyhistidine‐bearing polypeptides and proteins on attenuated total reflecting (ATR) sensor surfaces for the detection of biomolecular interactions by FTIR spectroscopy. Nitrilotriacetic acid (NTA) groups are covalently attached to self‐assembled monolayers of either thioalkanes on gold films or mercaptosilanes on silicon dioxide films deposited on germanium internal reflection elements. Complex formation between Ni²⁺ ions and NTA groups activates the ATR sensor surface for the selective binding of polyhistidine sequences. This approach not only allows a stable and reversible immobilization of histidine‐tagged peptides (His–peptides) but also simultaneously allows the direct in situ quantification of surface‐adsorbed molecules from their specific FTIR spectral bands. The surface concentrations of both NTA and His–peptide on silanized surfaces were determined to be 1.1 and 0.4 molecules nm^?2, respectively, which means that the surface is densely covered. A comparison of experimental FTIR spectra with simulated spectra reveals a surface‐enhancement effect of one order of magnitude for the gold surfaces. With the presented sensor surfaces, new ways are opened up to investigate, in situ and with high sensitivity and reproducibility, protein–ligand, protein–protein, protein–DNA interactions, and DNA hybridization by ATR–FTIR spectroscopy.     

Reversible Self‐Assembly of Carboxylated Peptide‐Functionalized Gold Nanoparticles Driven by Metal‐Ion Coordination

Carboxylated peptide‐functionalized gold nanoparticles (peptide‐GNPs) self‐assemble into two‐ and three‐dimensional nanostructures in the presence of various heavy metal ions (i.e. Pb²⁺, Cd²⁺, Cu²⁺, and Zn²⁺) in aqueous solution. The assembly process is monitored by following the changes in the surface plasmon resonance (SPR) band of gold nanoparticles in a UV/Vis spectrophotometer, which shows the development of a new SPR band in the higher‐wavelength region. The extent of assembly is dependent on the amount of metal ions present in the medium and also the time of assembly. TEM analysis clearly shows formation of two‐ and three‐dimensional nanostructures. The assembly process is completely reversible by addition of alkaline ethylenediaminetetraacetic acid (EDTA) solution. The driving force for the assembly of peptide‐GNPs is mainly metal ion/carboxylate coordination. The color and spectral changes due to this assembly can be used for detection of these heavy‐metal ions in solution.     

Self-assembled peptide tubelets with 7 A pores

Here we report the preparation and structural characteristics of self-assembling peptide tubelets composed of 32-membered rings formed of alternating alpha-amino acids and cis-3-aminocyclohexanecarboxylic acids. The tubelets possess a partial hydrophobic core environment, provided by the projection of the cyclohexane C2 methylene moiety into the lumen, and a Van der Waals pore diameter of about 7 A.     

Design of Amphiphilic Peptide Geometry towards Biomimetic Self‐Assembly of Chiral Mesoporous Silica

In nature, diatoms and sponges are exquisite examples of well‐defined structures produced by silica biomineralisation, in which proteins play an important role. However, the artificial peptide templating route for the silica mesostructure remains a formidable and unsolved challenge. Herein, we report our effort on the design of amphiphilic peptides for synthesising a highly ordered two‐dimensional (2D)‐hexagonal and lamellar chiral silica mesostructure using trimethoxysilylpropyl‐N,N,N‐trimethylammonium chloride as the co‐structure directing agent (CSDA). The geometry of the peptide was designed by adding proline residues into the hydrophobic chain of the peptide to break the β‐sheet conformation by weakening the intermolecular hydrogen bonds; this led to the mesophase transformation from the most general lamellar structure to the 2D hexagonal P6mm mesostructure by increasing the amphiphilic molecules packing parameter g. Enantiomerically pure chiral mesostructures were formed thanks to the intrinsic chirality and relatively strong intermolecular hydrogen bonds of peptides.