Polynucleotide transport through lipid membrane in the presence of starburst cyclodextrin-based poly(ethylene glycol)s.

Symmetrical cyclodextrin-based 14-arm star polymers with poly(ethylene glycol) PEG branches were synthesized and characterized. Interactions of the star polymers with lipid bilayers were studied by the "black lipid membrane" technique in order to demonstrate the formation of monomolecular artificial channels. The conditions for the insertion are mainly based on dimensions and amphiphilic properties of the star polymers, in particular the molar mass of the water-soluble polymer branches. Translocation of single-strand DNA (ssDNA) through those synthetic nanopores was investigated, and the close dimension between the cross-section of ssDNA and the cyclodextrin cavity led to an energy barrier that slowed down the translocation process.

From current trace to the understanding of confined media.

Nanopores constitute devices for the sensing of nano-objects such as ions, polymer chains, proteins or nanoparticles. We describe what information we can extract from the current trace. We consider the entrance of polydisperse chains into the nanopore, which leads to a conductance drop. We describe the detection of these current blockades according to their shape. Finally, we explain how data analysis can be used to enhance our understanding of physical processes in confined media.

Dynamics of a polyelectrolyte through aerolysin channel as a function of applied voltage and concentration⋆.

We describe the behaviour of a polyelectrolyte in confined geometry. The transport of a polyelectrolyte, dextran sulfate, through a recombinant protein channel, aerolysin, inserted into a planar lipid bilayer is studied as a function of applied voltage and polyelectrolyte concentration and chain length. The aerolysin pore has a weak geometry asymmetry, a high number of charged residues and the polyelectrolyte is strongly negatively charged. The resulting current blockades were characterized by short and long dwelling times. Their frequency varies exponentially as a function of applied voltage and linearly as a function of polyelectrolyte concentration. The long blockade duration decreases exponentially when the electrical force increases. The ratio of the population of short events to the one of long events decreases when the applied voltage increases and displays an exponential variation. The long residence time increases with the polyelectrolyte chain length. We measure a reduction of the effective charge of the polyelectrolyte at the pore entry and inside the channel. For a fixed applied voltage, + / - 100 mV, at both sides of the protein pore entrance, the events frequency is similar as a function of dextran sulfate concentration. The mean blockade durations are independent of polyelectrolyte concentration and are similar for both entrances of the pore and remain constant as a function of the electrical force.

Uncooked spaghetti in a colander: Injection of semiflexible polymers in a nanopore.

We study the flow injection of semiflexible polymers in a nanopore with a diameter smaller than the persistence length of the macromolecules. The suction model from de Gennes and Brochard is modified to take into account the effect of the rigidity of the polymer in the Odijk regime. We show that in this case of extreme confinement the flow threshold vanishes slowly and that in the limit of infinitely small nanopore the free energy barrier eventually disappears.

Nuclear pore complex plasticity during developmental process as revealed by super-resolution microscopy.

Nuclear Pore Complex (NPC) is of paramount importance for cellular processes since it is the unique gateway for molecular exchange through the nucleus. Unraveling the modifications of the NPC structure in response to physiological cues, also called nuclear pore plasticity, is key to the understanding of the selectivity of this molecular machinery. As a step towards this goal, we use the optical super-resolution microscopy method called direct Stochastic Optical Reconstruction Microscopy (dSTORM), to analyze oocyte development impact on the internal structure and large-scale organization of the NPC. Staining of the FG-Nups proteins and the gp210 proteins allowed us to pinpoint a decrease of the global diameter by measuring the mean diameter of the central channel and the luminal ring of the NPC via autocorrelation image processing. Moreover, by using an angular and radial density function we show that development of the Xenopus laevis oocyte is correlated with a progressive decrease of the density of NPC and an ordering on a square lattice.

Temperature Effect on Ionic Current and ssDNA Transport through Nanopores.

We have investigated the role of electrostatic interactions in the transport of nucleic acids and ions through nanopores. The passage of DNA through nanopores has so far been conjectured to involve a free-energy barrier for entry, followed by a downhill translocation where the driving voltage accelerates the polymer. We have tested the validity of this conjecture by using two toxins, α-hemolysin and aerolysin, which differ in their shape, size, and charge. The characteristic timescales in each toxin as a function of temperature show that the entry barrier is ∼15 kBT and the translocation barrier is ∼35 kBT, although the electrical force in the latter step is much stronger. Resolution of this fact, using a theoretical model, reveals that the attraction between DNA and the charges inside the barrel of the pore is the most dominant factor in determining the translocation speed and not merely the driving electrochemical potential gradient.

Evidence of unfolded protein translocation through a protein nanopore.

Protein nanopores are mainly used to study transport, unfolding, intrinsically disordered proteins, protein-pore interactions, and protein-ligand complexes. This single-molecule sensor for biomedical and biotechnological applications is promising but until now direct proof of protein translocation through a narrow channel is lacking. Here, we report the translocation of a chimera molecule through the aerolysin nanopore in the presence of a denaturing agent, guanidium chloride (1.5 M) and KCl (1 M). The chimera molecule is composed of the recombinant MalE protein with a unique cysteine residue at the C-terminal position covalently linked to a single-stranded DNA oligonucleotide. Real-time polymerase chain reaction (PCR) was used to detect the presence of chimera molecules that have been effectively translocated from the cis to trans chamber of the set up. Comparing the electrical signature of the chimera related to the protein or oligonucleotide alone demonstrates that each type of molecule displays different dynamics in term of transport time, event frequency, and current blockade. This original approach provides the possibility to study protein translocation through different biological, artificial, and biomimetic nanopores or nanotubes. New future applications are now conceivable such as protein refolding at the nanopore exit, peptides and protein sequencing, and peptide characterization for diagnostics.

Zero-mode waveguide detection of flow-driven DNA translocation through nanopores.

We directly measure the flow-driven injection of DNA through nanopores at the level of single molecule and single pore using a modified zero-mode waveguide method. We observe a flow threshold independent of the pore radius, the DNA concentration, and length. We demonstrate that the flow injection of DNA in nanopores is controlled by an energy barrier as proposed in the de Gennes-Brochard suction model. Finally, we show that the height of the energy barrier is modulated by functionalizing the nanopores.

Protein unfolding through nanopores.

In this mini-review we introduce and discuss a new method, at single molecule level, to study the protein folding and protein stability, with a nanopore coupled to an electric detection. Proteins unfolded or partially folded passing through one channel submitted to an electric field, in the presence of salt solution, induce different detectable blockades of ionic current. Their duration depends on protein conformation. For different studies proteins through nanopores, completely unfolded proteins induce only short current blockades. Their frequency increases as the concentration of denaturing agent or temperature increases, following a sigmoidal denaturation curve. The geometry or the net charge of the nanopores does not alter the unfolding transition, sigmoidal unfolding curve and half denaturing concentration or half temperature denaturation. A destabilized protein induces a shift of the unfolding curve towards the lower values of the denaturant agent compared to the wild type protein.Partially folded proteins exhibit very long blockades in nanopores. The blockade duration decreases when the concentration of denaturing agent increases. The variation of these blockades could be associated to a possible glassy behaviour.

Kinetics of enzymatic degradation of high molecular weight polysaccharides through a nanopore: experiments and data-modeling.

The enzymatic degradation of long polysaccharide chains is monitored by nanopore detection. It follows a Michaelis-Menten mechanism. We measure the corresponding kinetic constants at the single molecule level. The simulation results of the degradation process allowed one to account for the oligosaccharide size distribution detected by a nanopore.

Single molecule detection of glycosaminoglycan hyaluronic acid oligosaccharides and depolymerization enzyme activity using a protein nanopore.

Glycosaminoglycans are biologically active anionic carbohydrates that are among the most challenging biopolymers with regards to their structural analysis and functional assessment. The potential of newly introduced biosensors using protein nanopores that have been mainly described for nucleic acids and protein analysis to date, has been here applied to this polysaccharide-based third class of bioactive biopolymer. This nanopore approach has been harnessed in this study to analyze the hyaluronic acid glycosamiglycan and its depolymerization-derived oligosaccharides. The translocation of a glycosaminoglycan is reported using aerolysin protein nanopore. Nanopore translocation of hyaluronic acid oligosaccharides was evidenced by the direct detection of translocated molecules accumulated into the arrival compartment using high-resolution mass spectrometry. Anionic oligosaccharides of various polymerization degrees were discriminated through measurement of the dwelling time and translocation frequency. This molecular sizing capability of the protein nanopore device allowed the real-time recording of the enzymatic cleavage of hyaluronic acid polysaccharide. The time-resolved detection of enzymatically produced oligosaccharides was carried out to monitor the depolymerization enzyme reaction at the single-molecule level.

Mapping the conformational stability of maltose binding protein at the residue scale using nuclear magnetic resonance hydrogen exchange experiments.

Being able to differentiate local fluctuations from global folding-unfolding dynamics of a protein is of major interest for improving our understanding of structure-function determinants. The maltose binding protein (MBP), a protein that belongs to the maltose transport system, has a structure composed of two globular domains separated by a rigid-body "hinge bending". Here we determined, by using hydrogen exchange (HX) nuclear magnetic resonance experiments, the apparent stabilization free energies of 101 residues of MBP bound to ß-cyclodextrin (MBP-ßCD) under native conditions. We observed that the last helix of MBP (helix α14) has a lower protection factor than the rest of the protein. Further, HX experiments were performed using guanidine hydrochloride under subdenaturing conditions to discriminate between local fluctuations and global unfolding events and to determine the MBP-ßCD energy landscape. The results show that helix α4 and a part of helices α5 and α6 are clearly grouped into a subdenaturing folding unit and represent a partially folded intermediate under native conditions. In addition, we observed that amide protons located in the hinge between the two globular domains share similar ΔG(gu)(app) and m values and should unfold simultaneously. These observations provide new points of view for improving our understanding of the thermodynamic stability and the mechanisms that drive folding-unfolding dynamics of proteins.

Protein transport through a narrow solid-state nanopore at high voltage: experiments and theory.

We report experimentally the transport of an unfolded protein through a narrow solid-state nanopore of 3 nm diameter as a function of applied voltage. The random coil polypeptide chain is larger than the nanopore. The event frequency dependency of current blockades from 200 to 750 mV follows a van't Hoff-Arrhenius law due to the confinement of the unfolded chain. The protein is an extended conformation inside the pore at high voltage. We observe that the protein dwell time decreases exponentially at medium voltage and is inversely proportional to voltage for higher values. This is consistent with the translocation mechanism where the protein is confined in the pore, creating an entropic barrier, followed by electrophoretic transport. We compare these results to our previous work with a larger pore of 20 nm diameter. Our data suggest that electro-osmotic flow and protein adsorption on the narrowest nanopore wall are minimized. We discuss the experimental data obtained as compared with recent theory for the polyelectrolyte translocation process. This theory reproduces clearly the experimental crossover between the entropic barrier regime with medium voltage and the electrophoretic regime with higher voltage.

DNA unzipping and protein unfolding using nanopores.

We present here an overview on unfolding of biomolecular structures as DNA double strands or protein folds. After some theoretical considerations giving orders of magnitude about transport timescales through pores, forces involved in unzipping processes … we present our experiments on DNA unzipping or protein unfolding using a nanopore. We point out the difficulties that can be encountered during these experiments, such as the signal analysis problems, noise issues, or experimental limitations of such system.

Thermal unfolding of proteins probed at the single molecule level using nanopores.

The nanopore technique has great potential to discriminate conformations of proteins. It is a very interesting system to mimic and understand the process of translocation of biomacromolecules through a cellular membrane. In particular, the unfolding and folding of proteins before and after going through the nanopore are not well understood. We study the thermal unfolding of a protein, probed by two protein nanopores: aerolysin and α-hemolysin. At room temperature, the native folded protein does not enter into the pore. When we increase the temperature from 25 to 50 °C, the molecules unfold and the event frequency of current blockade increases. A similar sigmoid function fits the normalized event frequency evolution for both nanopores, thus the unfolding curve does not depend on the structure and the net charge of the nanopore. We performed also a circular dichroism bulk experiment. We obtain the same melting temperature (around 45 °C) using the bulk and single molecule techniques.

Transport of long neutral polymers in the semidilute regime through a protein nanopore.

We investigate the entrance of single poly(ethylene glycol) chains into an α-hemolysin channel. We detect the frequency and duration of the current blockades induced by large neutral polymers, where chain radius is larger than pore diameter. In the semidilute regime, these chains pass only if the monomer concentration is larger than a well-defined threshold. Experiments are performed in a very large domain of concentration and molecular mass, up to 35% and 200 kDa, respectively, which was previously unexplored. The variation of the dwell time as a function of molecular mass shows that the chains are extracted from the semidilute solution in contact with the pore by a reptation mechanism.

Wild type, mutant protein unfolding and phase transition detected by single-nanopore recording.

Understanding protein folding remains a challenge. A difficulty is to investigate experimentally all the conformations in the energy landscape. Only single molecule methods, fluorescence and force spectroscopy, allow observing individual molecules along their folding pathway. Here we observe that single-nanopore recording can be used as a new single molecule method to explore the unfolding transition and to examine the conformational space of native or variant proteins. We show that we can distinguish unfolded states from partially folded ones with the aerolysin pore. The unfolding transition curves of the destabilized variant are shifted toward the lower values of the denaturant agent compared to the wild type protein. The dynamics of the partially unfolded wild type protein follows a first-order transition. The denaturation curve obtained with the aerolysin pore is similar to that obtained with the α-hemolysin pore. The nanopore geometry or net charge does not influence the folding transition but changes the dynamics.

Phase behavior and structure of stable complexes between a long polyanion and a branched polycation.

The association between oppositely charged branched polyethylenimine (BPEI) and polymethacrylic acid (PMA) in the dilute regime is investigated using turbidimetric titration and electrophoretic mobility measurements. The complexation is controlled by tuning continuously the pH-sensitive charge of the polyacid in acidic solution. The formation of soluble and stable positively charged complexes is a cooperative process characterized by the existence of two regimes of weak and strong complexation. In the regime of weak complexation, a long PMA chain overcharged by several BPEI molecules forms a binary complex. As the charge of the polyacid increases, these binary complexes condense at a well defined charge ratio of the mixture to form large positively charged aggregates. The overcharging and the existence of two regimes of complexation are analyzed in the light of recent theories. The structure of the polyelectrolytes is investigated at higher polymer concentration by small angle neutron scattering. Binary complexes of finite size present an open structure where the polyacid chains connecting a small number of BPEI molecules have shrunk slightly. In the condensed complexes, BPEI molecules, wrapped by polyacid chains, form networks of stretched necklaces.

Discrimination of neutral oligosaccharides through a nanopore.

The detection of oligosaccharides at the single-molecule level was investigated using a protein nanopore device. Neutral oligosaccharides of various molecular weights were translocated through a single α-hemolysin nanopore and their nano-transit recorded at the single-molecule level. The translocation of maltose and dextran oligosaccharides featured by 1→4 and 1→6 glycosidic bonds respectively was studied in an attempt to discriminate oligosaccharides according to their polymerization degree and glycosidic linkages. Oligosaccharides were translocated through a free diffusion regime indicating that they adopted an extended conformation during their translocation in the nanopore. The dwell time increased with molecular mass, suggesting the usefulness of nanopore as a molecular sizing device.