Probing DNA-lipid membrane interactions with a lipopeptide nanopore.

Association of DNA molecules with lipid bilayer membranes is of considerable interest for a large variety of applications in biotechnology. Here we introduce syringomycin E (SRE), a small pore-forming lipopeptide produced by the bacterium Pseudomonas syringae, as a facile sensor for the detection of DNA interactions with lipid membranes. SRE forms highly reproducible pores in cellular and artificial membranes. The pore structure involves bilayer lipids, which have a pronounced influence on open channel conductance and gating. SRE channels act as ionic diodes that serve as current rectifiers sensitive to the charge of the bilayer. We employ this intrinsic property to electronically monitor the association of DNA molecules with the membrane in a variety of different settings. We show that SRE can be used for quantitatively probing electrostatic interactions of DNA and DNA-cholesterol conjugates with a lipid membrane. Furthermore, we demonstrate that SRE channels allow monitoring of hybridization reactions between lipid-anchored probe strands and complementary strands in solution. In the presence of double-stranded DNA, SRE channels display a particularly high degree of rectification. Finally, the formation of multilayered structures assembled from poly-(L)-lysine and DNA oligonucleotides on the membrane was precisely monitored with SRE.

Toward screening for antibiotics with enhanced permeation properties through bacterial porins.

Gram-negative bacteria are protected by an outer membrane barrier, and to reach their periplasmic target, penicillins have to diffuse through outer membrane porins such as OmpF. Here we propose a structure-dynamics-based strategy for improving such antibiotic uptake. Using a variety of experiments (high-resolution single channel recording, Minimum Inhibitory Concentration (MIC), liposome swelling assay) and accelerated molecular simulations, we decipher the subtle balance of interactions governing ampicillin diffusion through the porin OmpF. This suggests mutagenesis of a hot spot residue of OmpF for which additional simulations reveal drastic changes in the molecular and energetic pathway of ampicillin's diffusion. Inverting the problem, we predict and describe how benzylpenicillin diffuses with a lower effective energy barrier by interacting differently with OmpF. The thorough comparison between the theoretical predictions and the three independent experiments, which were set up to measure the kinetics of transport and biological activity, gives insights on how to combine such different investigation techniques with the aim of providing complementary validation. Our study illustrates the importance of microscopic interactions at the constriction region of the biological channel to control the antibiotic flux through it. We conclude by providing a complete inventory of the channel and antibiotic hot spots and discuss the implications in terms of antibacterial screening and design.

Bridging timescales and length scales: from macroscopic flux to the molecular mechanism of antibiotic diffusion through porins.

Our aim in this study was to provide an atomic description of ampicillin translocation through OmpF, the major outer membrane channel in Escherichia coli and main entry point for beta-lactam antibiotics. By applying metadynamics simulations, we also obtained the energy barriers along the diffusion pathway. We then studied the effect of mutations that affect the charge and size at the channel constriction zone, and found that in comparison to the wild-type, much lower energy barriers are required for translocation. The expected higher translocation rates were confirmed on the macroscopic scale by liposome-swelling assays. A microscopic view on the millisecond timescale was obtained by analysis of temperature-dependent ion current fluctuations in the presence of ampicillin and provide the enthalpic part of the energy barrier. By studying antibiotic translocation over various timescales and length scales, we were able to discern its molecular mechanism and rate-limiting interactions, and draw biologically relevant conclusions that may help in the design of drugs with enhanced permeation rates.

Sequence-dependent unfolding kinetics of DNA hairpins studied by nanopore force spectroscopy.

Nanopore force spectroscopy is used to study the unzipping kinetics of two DNA hairpin molecules with a 12 base pair long stem containing two contiguous stretches of six GC and six AT base pairs in interchanged order. Even though the thermodynamic stabilities of the two structures are nearly the same, they differ greatly in their unzipping kinetics. When the GC segment has to be broken before the AT segment, the unfolding rate is orders of magnitude smaller than in the opposite case. We also investigated hairpins with stem regions consisting only of AT or GC base pairs. The pure AT hairpins translocate much faster than the other hairpins, whereas the pure GC hairpins translocate on similar timescales to the hairpins with only an initial GC segment. For each hairpin, nanopore force spectroscopy is performed for different loading rates and the resulting unzipping distributions are mathematically transformed to a master curve that yields the unfolding rate as a function of applied voltage. This is compared with a stochastic model of the unfolding process for the two sequences for different voltages. The results can be rationalized in terms of the different natures of the free energy landscapes for the unfolding process.

Understanding ion conductance on a molecular level: an all-atom modeling of the bacterial porin OmpF.

All-atom molecular dynamics simulations of the ion current through OmpF, the major porin in the outer membrane of Escherichia coli, were performed. Starting from the crystal structure, the all-atom modeling allows us to calculate a parameter-free ion conductance in semiquantitative agreement with experiment. Discrepancies between modeling and experiment occur, e.g., at salt concentrations above 1 M KCl or at high temperatures. At lower salt concentrations, the ions have separate pathways along the channel surface. The constriction zone in the channel contains, on one side, a series of positively charges (R42, R82, R132), and on the opposite side, two negatively charged residues (D113, E117). Mutations generated in the constriction zone by removing cationic residues enhance the otherwise small cation selectivity, whereas removing the anionic residues reverses the selectivity. Reduction of the negatively charged residues decreases the conductance by half, whereas cationic residues enhance the conductance. Experiments on mutants confirm the results of the molecular-level simulations.

How beta-lactam antibiotics enter bacteria: a dialogue with the porins.

BACKGROUND: Multi-drug resistant (MDR) infections have become a major concern in hospitals worldwide. This study investigates membrane translocation, which is the first step required for drug action on internal bacterial targets. beta-lactams, a major antibiotic class, use porins to pass through the outer membrane barrier of Gram-negative bacteria. Clinical reports have linked the MDR phenotype to altered membrane permeability including porin modification and efflux pump expression. METHODOLOGY/PRINCIPAL FINDINGS: Here influx of beta-lactams through the major Enterobacter aerogenes porin Omp36 is characterized. Conductance measurements through a single Omp36 trimer reconstituted into a planar lipid bilayer allowed us to count the passage of single beta-lactam molecules. Statistical analysis of each transport event yielded the kinetic parameters of antibiotic travel through Omp36 and distinguishable translocation properties of beta-lactams were quantified for ertapenem and cefepime. Expression of Omp36 in an otherwise porin-null bacterial strain is shown to confer increases in the killing rate of these antibiotics and in the corresponding bacterial susceptibility. CONCLUSIONS/SIGNIFICANCE: We propose the idea of a molecular "passport" that allows rapid transport of substrates through porins. Deciphering antibiotic translocation provides new insights for the design of novel drugs that may be highly effective at passing through the porin constriction zone. Such data may hold the key for the next generation of antibiotics capable of rapid intracellular accumulation to circumvent the further development MDR infections.

Altering the activity of syringomycin E via the membrane dipole potential.

The membrane dipole potential is responsible for the modulation of numerous biological processes. It was previously shown (Ostroumova, O. S.; Kaulin, Y. A.; Gurnev, P. A.; Schagina, L. V. Langmuir 2007, 23, 6889-6892) that variations in the dipole potential lead to changes in the channel properties of the antifungal lipodepsipeptide syringomycin E (SRE). Here, data are presented demonstrating the effect of the membrane dipole potential on the channel-forming activity of SRE. A rise in the dipole potential is accompanied by both an increase in the minimum SRE concentration required for the detection of single channels at fixed voltage and a decrease in the steady-state number of open SRE channels at a given SRE concentration and voltage. These alterations are determined by several factors: gating charge, connected with translocations of lipid and SRE dipoles during channel formation, the bilayer-water solution partitioning of SRE, and the chemical work related to conformational changes during channel formation.

Actin and amphiphilic polymers influence on channel formation by Syringomycin E in lipid bilayers.

The bacterial lipodepsipeptide syringomycin E (SRE) added to one (cis-) side of bilayer lipid membrane forms voltage dependent ion channels. It was found that G-actin increased the SRE-induced membrane conductance due to formation of additional SRE-channels only in the case when actin and SRE were applied to opposite sides of a lipid bilayer. The time course of conductance relaxation depended on the sequence of SRE and actin addition, suggesting that actin binds to the lipid bilayer and binding is a limiting step for SRE-channel formation. G-actin adsorption on the membrane was irreversible. The amphiphilic polymers, Konig's polyanion (KP) and poly(Lys, Trp) (PLT) produced the actin-like effect. It was shown that the increase in the SRE membrane activity was due to hydrophobic interactions between the adsorbing molecules and membrane. Nevertheless, hydrophobic interactions were not sufficient for the increase of SRE channel-forming activity. The dependence of the number of SRE-channels on the concentration of adsorbing species gave an S-shaped curve indicating cooperative adsorption of the species. Kinetic analysis of SRE-channel number growth led to the conclusion that the actin, KP, and PLT molecules form aggregates (domains) on the trans-monolayer. It is suggested that an excess of SRE-channel formation occurs within the regions of the cis-monolayer adjacent to the domains of the adsorbed molecules, which increase the effective concentration of SRE-channel precursors.