Transport de macromolécules à travers un pore nanométrique unique

Nous présentons dans ce travail une étude expérimentale du transport de différents polymères, neutres, chargés, et partiellement structurés à travers un pore protéique nanométique (l -Hémolysine du Staphylocoque doré). Dans un domaine où la taille des chaînes de polymères est comparable ou supérieure au diamètre du pore. Le pore est inséré dans une bicouche lipidique plane soumise à une différence de potentiel. Le passage de polymère est détecté électriquement en mesurant les fluctuations du courant électrique induite par la présence des chaînes dans le pore. Nous avons mis en évidence un seuil de concentration en régime semi-dilué permettant le passage de grandes chaînes neutres et flexibles de taille très supérieure devant le diamètre du pore. Le temps de passage de ces chaînes obtenu expérimentalement est en excellent accord avec celui décrit par le modèle de reptation. Cependant la dynamique des chaînes de taille comparable au diamètre du pore est anormalement ralentie comparée aux différentes prédictions théoriques. Nous avons prouvé expérimentalement l existence d un seuil en force ionique qui gouverne l entrée des chaînes chargées. Une chaîne chargée passe dans le pore seulement si l épaisseur du nuage de contre ions qui entour la chaîne est inférieure au diamètre du pore. Nous avons mis en évidence l existence d états intermédiaires correspondant à longue durée de vie dans des protéine partiellement repliées. De plus nous avons montré la coexistence de conformation dépliée et partiellement repliée pour une concentration donnée en agent dénaturant.An experimental study of the transport of various polymers chains, neutral, charged, and partially structured through a proteic nanopore ( -Hémolysin from Staphylococcus aureus) is presented in this work. In the domain where the size of the polymer chains is comparable or higher than the diameter of the pore. A proteinic pore is inserted in lipid bilayers subjected to an electrical tension. The passage of chains is detected electrically by measuring the electrical current fluctuation induced by the presence of chains in the pore. We observe a threshold of concentration in the semi-dilute regime allowing the passage of large neural flexible chains. The residence time of these chains obtained in experiments is in excellent agreement with that described by the model of reptation . However the dynamics of the chains of size comparable with the diameter of the pore is abnormally slowed down compared with the various theoretical predictions. We proved in experiments the existence of a threshold in ionic strength which controls the entry of the charged chains. A charged chain passes in the pore only if the thickness of the cloud of conter-ions around the chain is smaller than the diameter of the pore. We highlighted the existence of intermediate states corresponding to long life in protein partially folded. Moreover we showed the coexistence of conformation (unfolded and partially folded) for a given concentration of denaturing agent.EVRY-BU (912282101) / SudocSudocFranceF

Focus on Protein Unfolding Through Nanopores

International audienceIn this review, we focus only on the unfolding of proteins through nanopores. We introduce the principle of electrical detection with nanopores and how this technique provides information using an electric signal. We describe different pioneer studies on protein unfolding through protein channels and through solid-state nanopores. We discuss different methods to study protein unfolding at the single-molecule level and the advantages this new nanopore technique offers

Electrophoresis and Electroosmosis in Aerolysin and Hemolysin Nanopores

Voltage-dependent interaction of macromolecules with biological nanopores can arise from electroosmotic (EOF) or electrophoretic (EPF) forces. Both poly(ethylene-glycol) (PEG), and cyclodextrins (β- and α-CD, respectively) block the ionic current through α-hemolysin (αHL) and Aerolysin (AeL) pore-forming proteins with well-documented voltage-dependence. Here, we attempt to relate differential effects of the electrolytes KCl and LiCl on the voltage-dependence of frequency and dwell-time of blocks to the relative contributions of EOF and EPF.For AeL, irrespective of electrolyte, blocks by α-CD only occurred from the pore's cis-side and with trans-positive voltages. Blocks in LiCl were much longer than those in KCl (11ms vs. 760μs at 90mV) but dwell times showed a positive, linear voltage dependence in both cases (LiCl:6fold/100mV; KCl:3fold/100mV). In KCl, cis-side PEG blocked AeL with trans-negative voltages only.1 Surprisingly, in LiCl, blocks occurred only at trans-positive voltages and at more than 1000 fold PEG-concentration but we still are in a very diluted regime. Blocks were shorter than in KCl by a factor of 5 (60 vs. 320μs at 100mV), but frequency (3fold/100mV) and dwell time (2fold/100mV) depended linearly on voltage, reminiscent of the behavior of α-CD in both electrolytes.Linear voltage dependence of block frequency might indicate dominance of EOF for pore entry of PEG in LiCl and for α-CD generally, while an exponential voltage-dependence would be expected for an electrophoretic force. Indeed, for PEG in KCl, which may be positively charged due to K+-chelation (see Ref.1 for discussion), we found an exponential increase in frequency for blocks of AeL and αHL with increasing trans-negative and trans-positive voltages, respectively. We cannot, however, exclude voltage and ion-dependent effects on the binding reaction, which, rather than partitioning, may be rate-limiting for the electrophysiologically detectable blockages

High-Resolution Size-Discrimination of Single Nonionic Synthetic Polymers with a Highly Charged Biological Nanopore

International audienceElectrophysiological studies of the interaction of polymers with pores formed by bacterial toxins (1) provide a window on single molecule interaction with proteins in real time, (2) report on the behavior of macromolecules in confinement, and (3) enable label-free single molecule sensing. Using pores formed by the staphylococcal toxin α-hemolysin (aHL), a particularly pertinent observation was that, under high salt conditions (3-4 M KCl), the current through the pore is blocked for periods of hundreds of microseconds to milliseconds by poly(ethylene glycol) (PEG) oligomers (degree of polymerization approximately 10-60). Notably, this block showed monomeric sensitivity on the degree of polymerization of individual oligomers, allowing the construction of size or mass spectra from the residual current values. Here, we show that the current through the pore formed by aerolysin (AeL) from Aeromonas hydrophila is also blocked by PEG but with drastic differences in the voltage-dependence of the interaction. In contrast to aHL, AeL strongly binds PEG at high transmembrane voltages. This fact, which is likely related to AeL's highly charged pore wall, allows discrimination of polymer sizes with particularly high resolution. Multiple applications are now conceivable with this pore to screen various nonionic or charged polymers

Probing driving forces in aerolysin and α-hemolysin biological nanopores: electrophoresis versus electroosmosis

International audienceThe transport of macromolecules through nanopores is involved in many biological functions and is today at the basis of promising technological applications. Nevertheless the interpretation of the dynamics of the macromolecule/nanopore interaction is still misunderstood and under debate. At the nanoscale, inside biomimetic channels under an external applied voltage, electrophoresis, which is the electric force acting on electrically charged molecules, and electroosmotic flow (EOF), which is the fluid transport associated with ions, contribute to the direction and magnitude of the molecular transport. In order to decipher the contribution of the electrophoresis and electroosmotic flow, we explored the interaction of small, rigid, neutral molecules (cyclodextrins) and flexible, non-ionic polymers (poly(ethylene glycol), PEG) that can coordinate cations under appropriate experimental conditions, with two biological nanopores: aerolysin (AeL) and α-hemolysin (aHL). We performed experiments using two electrolytes with different ionic hydration (KCl and LiCl). Regardless of the nature of the nanopore and of the electrolyte, cyclodextrins behaved as neutral analytes. The dominant driving force was attributed to EOF, acting in the direction of the anion flow and stronger in LiCl than in KCl. The same qualitative behaviour was observed for PEGs in LiCl. In contrast, in KCl, PEGs behaved as positively charged polyelectrolytes through both AeL and aHL. Our results are in agreement with theoretical predictions about the injection of polymers inside a confined geometry (ESI). We believe our results to be of significant importance for better control of the dynamics of analytes of different nature through biological nanopores

Sensing Proteins through Nanopores: Fundamental to Applications

International audienceProteins subjected to an electric field and forced to pass through a nanopore induce blockades of ionic current that depend on the protein and nanopore characteristics and interactions between them. Recent advances in the analysis of these blockades have highlighted a variety of phenomena that can be used to study protein translocation and protein folding, to probe single-molecule catalytic reactions in order to obtain kinetic and thermodynamic information, and to detect protein–antibody complexes, proteins with DNA and RNA aptamers, and protein–pore interactions. Nanopore design is now well controlled, allowing the development of future biotechnologies and medicine applications

Discrimination of neutral oligosaccharides through a nanopore

International audienceThe 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

Exploration of Neutral Versus Polyelectrolyte Behavior of Poly(ethylene glycol)s in Alkali Ion Solutions using Single-Nanopore Recording

International audienceWe examine the effect of alkali ions (Li+, Na+, K+, Rb+, Cs+) on the partitioning of neutral and flexible poly(ethylene glycol) into the alpha-hemolysin (α-HL) nanopore for a large range of applied voltages at high salt concentration. The neutral polymer behaves as if charged, that is, the event frequency increases with applied voltage, and the residence times decrease with the electric force for all cations except Li+. In contrast, in the presence of LiCl, we find the classical partitioning behavior of neutral polymers, that is, the event frequency and the residence times are independent of the applied voltage. Assuming that lithium does not associate with PEG enabled us to quantify the relative magnitude of the entropic and enthalpic contribution to the free- energy barrier and the number of complexed cations using two different arguments; the first estimate is based on the balance of forces, and the second is found comparing the blockade ratio in the presence of LiCl (no complexed ions) to the blockade ratio of chains in the presence of the other salts (with complexed ions). This estimate is in agreement with recent simulations. These findings demonstrate that the nanopore could prove useful for the rapid probing of the capabilities of different neutral molecules to form complexes with different ions