RESUMEN
Mimicking and extending the gating properties of biological pores is of paramount interest for the fabrication of membranes that could be used in filtration or drug processing. Here, we build a selective and switchable nanopore for macromolecular cargo transport. Our approach exploits polymer graftings within artificial nanopores to control the translocation of biomolecules. To measure transport at the scale of individual biomolecules, we use fluorescence microscopy with a zero-mode waveguide set up. We show that grafting polymers that exhibit a lower critical solution temperature creates a toggle switch between an open and closed state of the nanopore depending on the temperature. We demonstrate tight control over the transport of DNA and viral capsids with a sharp transition (â¼1 °C) and present a simple physical model that predicts key features of this transition. Our approach provides the potential for controllable and responsive nanopores in a range of applications.
RESUMEN
The implementation of a reliable, rapid, inexpensive, and simple method for whole-proteome identification would greatly benefit cell biology research and clinical medicine. Proteins are currently identified by cleaving them with proteases, detecting the polypeptide fragments with mass spectrometry, and mapping the latter to sequences in genomic/proteomic databases. Here, we demonstrate that the polypeptide fragments can instead be detected and classified at the single-molecule limit using a nanometer-scale pore formed by the protein aerolysin. Specifically, three different water-soluble proteins treated with the same protease, trypsin, produce different polypeptide fragments defined by the degree by which the latter reduce the nanopore's ionic current. The fragments identified with the aerolysin nanopore are consistent with the predicted fragments that trypsin could produce.
Asunto(s)
Toxinas Bacterianas/química , Citocromos c/análisis , Muramidasa/análisis , Mioglobina/análisis , Nanoporos , Proteínas Citotóxicas Formadoras de Poros/química , Aeromonas hydrophila/química , Citocromos c/química , Proteínas Hemolisinas/química , Muramidasa/química , Mioglobina/química , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/química , Proteolisis , Proteómica , Tripsina/químicaRESUMEN
Seeking new tools for the analysis of glycosaminoglycans, we have compared the translocation of anionic oligosaccharides from hyaluronic acid using aerolysin and [Formula: see text]-hemolysin nanopores. We show that pores of similar channel length and diameter lead to distinct translocation behavior of the same macromolecules, due to different structural properties of the nanopores. When passing from the vestibule side of the nanopores, short hyaluronic acid oligosaccharides could be detected during their translocation across an aerolysin nanopore but not across an [Formula: see text]-hemolysin nanopore. We were however able to detect longer oligosaccharide fragments, resulting from the in situ enzymatic depolymerization of hyaluronic acid polysaccharides, with both nanopores, meaning that short oligosaccharides were crossing the [Formula: see text]-hemolysin nanopore with a speed too high to be detected. The translocation speed was an order of magnitude higher across [Formula: see text]-hemolysin compared to aerolysin. These results show that the choice of a nanopore to be used for resistive pulse sensing experiments should not rely only on the diameter of the channel but also on other parameters such as the charge repartition within the pore lumen.
Asunto(s)
Toxinas Bacterianas/química , Técnicas Biosensibles/métodos , Proteínas Hemolisinas/química , Ácido Hialurónico/análisis , Ácido Hialurónico/química , Nanoporos , Proteínas Citotóxicas Formadoras de Poros/química , PolimerizacionRESUMEN
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 â¼15 kBT and the translocation barrier is â¼35 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.
Asunto(s)
Transporte Biológico , ADN de Cadena Simple , Nanoporos , Electricidad Estática , Temperatura , Toxinas Bacterianas/toxicidad , Transporte Biológico/efectos de los fármacos , Proteínas Hemolisinas/toxicidad , Membranas Artificiales , Modelos Teóricos , Movimiento (Física) , Fosfatidilcolinas , Polímeros , Proteínas Citotóxicas Formadoras de Poros/toxicidadRESUMEN
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.
Asunto(s)
ADN/química , Análisis de Inyección de Flujo/métodos , Modelos Químicos , Nanoporos , Bacteriófago lambda/genética , Benzoxazoles/química , ADN Viral/química , Colorantes Fluorescentes/química , Sustancias Intercalantes/química , Compuestos de Quinolinio/química , Relación Estructura-Actividad , TermodinámicaRESUMEN
Protein nanopores have proven to be effective for single-molecule studies, particularly for single-stranded DNA (ssDNA) translocation. Previous experiments demonstrated their ability to distinguish differences in purine and pyrimidine bases and in the orientation of the ssDNA molecule inside nanopores. Unfortunately, the microscopic details of ssDNA translocation over experimental time scales, which are not accessible through all-atom molecular dynamics (MD), have yet to be examined. However, coarse-grained (CG) MD simulations enable systems to be simulated over longer characteristic times closer to experiments than all-atom MD. This paper studies ssDNA translocation through α-hemolysin nanopores exploiting steered MD using the MARTINI CG force field. The impacts of the sequence length, orientation inside the nanopore and DNA charges on translocation dynamics as well as the conformational dynamics of ssDNA during the translocation are explored. Our results highlight the efficacy of CG molecular dynamics in capturing the experimental properties of ssDNA translocation, including a wide distribution in translocation times per base. In particular, the phosphate charges of the DNA molecule are crucial in the translocation dynamics and impact the translocation rate. Additionally, the influence of the ssDNA molecule orientation on the translocation rate is explained by the conformational differences of ssDNA inside the nanopore during its translocation. Our study emphasizes the significance of obtaining sufficient statistics via CG MD, which can elucidate the great variety of translocation processes.
Asunto(s)
ADN de Cadena Simple , Proteínas Hemolisinas , Simulación de Dinámica Molecular , Nanoporos , ADN de Cadena Simple/química , ADN de Cadena Simple/metabolismo , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Conformación de Ácido NucleicoRESUMEN
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.
Asunto(s)
Proteínas de Unión a Maltosa/química , Pliegue de Proteína , Desplegamiento Proteico , Hidrógeno/metabolismo , Espectroscopía de Resonancia Magnética , Estructura Secundaria de Proteína , TermodinámicaRESUMEN
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.
Asunto(s)
Toxinas Bacterianas/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas Hemolisinas/química , Nanoporos , Proteínas de Unión Periplasmáticas/química , Proteínas Citotóxicas Formadoras de Poros/química , Desplegamiento Proteico , Dicroismo Circular , TemperaturaRESUMEN
In order to understand the physical processes of nanopore experiments at the molecular level, microscopic information from molecular dynamics is greatly needed. Coarse-grained models are a good alternative to classical all-atom models since they allow longer and faster simulations. We performed coarse-grained molecular dynamics of the ionic transport through the α-hemolysin protein nanopore, inserted into a lipid bilayer surrounded by solvent and ions. For this purpose, we used the MARTINI coarse-grained force field and its polarizable water solvent (PW). Moreover, the electric potential difference applied experimentally was mimicked by the application of an electric field to the system. We present, in this study, the results of 1.5 µs long-molecular dynamics simulations of 12 different systems for which different charged amino acids were neutralized, each of them in the presence of nine different electric fields ranging between ±0.04 V/nm (a total of around 100 simulations). We were able to observe several specific features of this pore, current asymmetry and anion selectivity, in agreement with previous studies and experiments, and we identified the charged amino acids responsible for these current behaviors, therefore validating our coarse-grain approach to study ionic transport through nanopores. We also propose a microscopic explanation of these ionic current features using ionic density maps.
RESUMEN
Glycosaminoglycans are highly anionic functional polysaccharides with information content in their structure that plays a major role in the communication between the cell and the extracellular environment. The study presented here reports the label-free detection and analysis of glycosaminoglycan molecules at the single molecule level using sensing by biological nanopore, thus addressing the need to decipher structural information in oligo- and polysaccharide sequences, which remains a major challenge for glycoscience. We demonstrate that a wild-type aerolysin nanopore can detect and characterize glycosaminoglycan oligosaccharides with various sulfate patterns, osidic bonds and epimers of uronic acid residues. Size discrimination of tetra- to icosasaccharides from heparin, chondroitin sulfate and dermatan sulfate was investigated and we show that different contents and distributions of sulfate groups can be detected. Remarkably, differences in α/ß anomerization and 1,4/1,3 osidic linkages can also be detected in heparosan and hyaluronic acid, as well as the subtle difference between the glucuronic/iduronic epimers in chondroitin and dermatan sulfate. Although, at this stage, discrimination of each of the constituent units of GAGs is not yet achieved at the single-molecule level, the resolution reached in this study is an essential step toward this ultimate goal.
Asunto(s)
Glicosaminoglicanos , Nanoporos , Sulfatos de Condroitina/química , Dermatán Sulfato/análisis , Dermatán Sulfato/química , Glicosaminoglicanos/química , Polisacáridos/análisis , SulfatosRESUMEN
It is reported that low concentration of amphiphilic triblock copolymers of pMeOx-b-pTHF-b-pMeOx structure (TBCPs) improves gene expression in skeletal muscle upon intramuscular co-injection with plasmid DNA. Physicochemical studies carried out to understand the involved mechanism show that a phase transition of TBCPs under their unimer state is induced when the temperature is elevated from 25 to 37 °C, the body temperature. Several lines of evidences suggest that TBCP insertion in a lipid bilayer causes enough lipid bilayer destabilization and even pore formation, a phenomenon heightened during the phase transition of TBCPs. Interestingly, this property allows DNA translocation across the lipid bilayer model. Overall, the results indicate that TBCPs exhibiting a phase transition at the body temperature is promising to favor in vivo pDNA translocation in skeletal muscle cells for gene therapy applications.
Asunto(s)
ADN , Músculo Esquelético/metabolismo , Plásmidos , Transfección , Animales , ADN/genética , ADN/farmacología , Femenino , Ratones , Plásmidos/genética , Plásmidos/farmacologíaRESUMEN
We present experimental and theoretical results on the voltage-dependent escape dynamics of DNA hairpins of different orientations threaded into an alpha -hemolysin channel. Using a coarse-grained formulation, we map the motion of the polymer in the pore to that of a biased single-particle random walk along the translocation coordinate. By fitting the escape probability distributions obtained from theory to experimental data, we extract the voltage-dependent diffusion constants and bias-induced velocities. Using our two-parameter theory, we obtain excellent agreement with experimentally measured escape time distributions. Further, we find that the ratio of mean escape times for hairpins of different orientations is strongly voltage dependent, with the ratio of 3' - to 5' -threaded DNA decreasing from approximately 1.7 to approximately 1 with increasing assisting voltages V(a) . We also find that our model describes 5' -threaded DNA escape extremely well, while providing inadequate fits for 3' escape. Finally, we find that the escape times for both orientations are equal for high assisting voltages, suggesting that the interactions of DNA with the alpha -hemolysin channel are both orientation and voltage dependent.
Asunto(s)
Biofisica/métodos , ADN/química , Proteínas Hemolisinas/química , Polímeros/química , Difusión , Modelos Químicos , Modelos Moleculares , Modelos Estadísticos , Conformación Molecular , Conformación de Ácido Nucleico , Probabilidad , Conformación Proteica , Electricidad Estática , Factores de TiempoRESUMEN
We report a study on the dynamics of latex polystyrene beads and of DNA molecules confined in two dimensions, using fluorescence video-microscopy. We particularly focus on the character of the confined objects (hard or soft) and on the nature of the confinement: liquid (in a soap film) or solid (between two glass plates). For weak confinements, whatever the nature of confinement, we observe that DNA molecules and latex beads behave very similarly: the tighter the confinement, the slower the diffusion with a good agreement with theory. For strong confinements between solid walls (thickness of confinement smaller than the bulk radius of gyration), DNA coils are not immobilized and still diffuse. We show in this case that the conformation of DNA chains is in good agreement with the predictions of De Gennes and Brochard (radius approximately e (-1/4), with e the confinement gap); on the other hand, we cannot really check the theoretical predictions for the diffusion coefficient. Interestingly, strong confinement of latex beads in a soap film leads to a anomalous slow diffusion, certainly associated with an additional viscous drag generated by the interfaces.
Asunto(s)
ADN/química , Microesferas , Poliestirenos/química , Solventes/química , Difusión , Vidrio/química , Microscopía Fluorescente , Tamaño de la Partícula , Permeabilidad , Jabones/química , Propiedades de Superficie , Factores de Tiempo , ViscosidadRESUMEN
The aim of this work is to study pore protein denaturation inside a lipid bilayer and to probe current asymmetry as a function of the channel conformation. We describe the urea denaturation of alpha-hemolysin channel and the channel formation of alpha-hemolysin monomer incubated with urea prior to insertion into a lipid bilayer. Analysis of single-channel recordings of current traces reveals a sigmoid curve of current intensity as a function of urea concentration. The normalized current asymmetry at 29+/-4% is observed between 0 and 3.56M concentrations and vanishes abruptly down to 0 concentration exceeds 4M. The loss of current asymmetry through alpha-hemolysin is due to the denaturation of the channel's cap. We also show that the alpha-hemolysin pore inserted into a lipid bilayer is much more resistant to urea denaturation than the alpha-hemolysin monomer in solution: The pore remains in the lipid bilayer up to 7.2M urea. The pore formation is possible up to 4.66M urea when protein monomers were previously incubated in urea.
Asunto(s)
Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Membrana Dobles de Lípidos/química , Nanoestructuras/química , Urea/farmacología , Desnaturalización Proteica/efectos de los fármacosRESUMEN
We report an alternative method for electrophoretic separation of large DNAs using steric confinement between solid walls, without gel or obstacles. The change of electrophoretic mobility vs confinement thickness is investigated using fluorescence video microscopy. We observe separation at small confinement thicknesses followed by a transition to the bulk behavior (no separation) at a thickness of about 4 mum (a few radii of gyration for the studied DNA chains). We present tentative explanations of our original observations.
Asunto(s)
ADN/química , Campos Electromagnéticos , Electroforesis Capilar/métodos , Microscopía Fluorescente/métodos , Concentración Osmolar , Sensibilidad y EspecificidadRESUMEN
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.
Asunto(s)
Nanoporos , Desplegamiento Proteico , Proteínas/química , Animales , Biofisica/métodos , Humanos , Modelos Moleculares , Nanoporos/ultraestructura , Conformación Proteica , Desnaturalización Proteica , Estabilidad ProteicaRESUMEN
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.
Asunto(s)
Nanoporos , Desnaturalización de Ácido Nucleico , Desplegamiento Proteico , Algoritmos , Proteínas Bacterianas/química , Transporte Biológico , Electroósmosis , Proteínas Hemolisinas/química , Proteínas de Unión a Maltosa/química , Membranas ArtificialesRESUMEN
A striking feature of the alpha-hemolysin channel-a prime candidate for biotechnological applications-is the dependence of its ionic conductance on the magnitude and direction of the applied bias. Through a combination of lipid bilayer single-channel recording and molecular dynamics (MD) simulations, we characterized the current-voltage relationship of alpha-hemolysin for all alkali chloride salts at neutral pH. The rectification of the ionic current was found to depend on the type of cations and increase from Li(+) to Cs(+). Analysis of the MD trajectories yielded a simple quantitative model that related the ionic current to the electrostatic potential, the concentration and effective mobility of ions in the channel. MD simulations reveal that the major contribution to the current asymmetry and rectification properties originates from the cationic contribution to the current that is significantly reduced in a cationic dependent way when the membrane polarity is reversed. The variation of chloride current was found to be less important. We report that the differential affinity of cations for the charged residues positioned at the channel's end modulates the number of ions inside the channel stem thus affecting the current properties. Through direct comparison of simulation and experiment, this study evaluates the accuracy of the MD method for prediction of the asymmetric, voltage dependent conductances of a membrane channel.
RESUMEN
The time-dependent response of individual biomolecular complexes to an applied force can reveal their mechanical properties, interactions with other biomolecules, and self-interactions. In the past decade, a number of single-molecule methods have been developed and applied to a broad range of biological systems, such as nucleic acid complexes, enzymes and proteins in the skeletal and cardiac muscle sarcomere. Nanopore force spectroscopy (NFS) is an emerging single-molecule method, which takes advantage of the native electrical charge of biomolecule to exert a localized bond-rupture force and measure the biomolecule response. Here, we review the basic principles of the method and discuss two bond breakage modes utilizing either a fixed voltage or a steady voltage ramp. We describe a unified theoretical formalism to extract kinetic information from the NFS data, and illustrate the utility of this formalism by analyzing data from nanopore unzipping of individual DNA hairpin molecules, where the two bond breakage modes were applied.