Controlling a single protein in a nanopore through electrostatic traps

Protein-protein pore interaction is a fundamental and ubiquitous process in biology and medical biotechnology. Here, we employed high-resolution time-resolved single-channel electrical recording along with protein engineering to examine a protein-protein pore interaction at single-molecule resolution. The pore was formed by Staphylococcus aureus alpha-hemolysin (alphaHL) protein and contained electrostatic traps formed by rings of seven aspartic acid residues placed at two different positions within the pore lumen. The protein analytes were positively charged presequences (pb2) of varying length fused to the small ribonuclease barnase (Ba). The presence of the electrostatic traps greatly enhanced the interaction of the pb2-Ba protein with the alphaHL protein pore. This study demonstrates the high sensitivity of the nanopore technique to an array of factors that govern the protein-protein pore interaction, including the length of the pb2 presequence, the position of the electrostatic traps within the pore lumen, the ionic strength of the aqueous phase, and the transmembrane potential. Alterations in the functional properties of the pb2-Ba protein and the alphaHL protein pore and systematic changes of the experimental parameters revealed the balance between forces driving the pb2-Ba protein into the pore and forces driving it out.     

Temperature-responsive protein pores

We describe temperature-responsive protein pores containing single elastin-like polypeptide (ELP) loops. The ELP loops were placed within the cavity of the lumen of the alpha-hemolysin (alphaHL) pore, a heptamer of known crystal structure. The cavity is roughly spherical with a molecular surface volume of about 39,500 A3. In an applied potential, the wild-type alphaHL pore remained open for long periods. In contrast, the ELP loop-containing alphaHL pores exhibited transient current blockades, the nature of which depended on the length and sequence of the inserted loop. Together with similar results obtained with poly(ethylene glycols) covalently attached within the cavity, the data suggest that the transient current blockades are caused by excursions of ELP into the transmembrane beta-barrel domain of the pore. Below its transition temperature, the ELP loop is fully expanded and blocks the pore completely, but reversibly. Above its transition temperature, the ELP is dehydrated and the structure collapses, enabling a substantial flow of ions. Potential applications of temperature-responsive protein pores in medical biotechnology are discussed.     

Protein nanopores with covalently attached molecular adapters

Molecular adapters are crucial for the stochastic sensing of organic analytes with alpha-hemolysin (alphaHL) protein nanopores when direct interactions between analytes and the pore cannot readily be arranged by conventional protein engineering. In our earlier studies, cyclodextrin adapters were lodged noncovalently within the lumen of the alphaHL pore. In the present work, we have realized the controlled covalent attachment of a beta-cyclodextrin (betaCD) adapter in the two possible molecular orientations inside alphaHL pores prepared by genetic engineering. There are two advantages to such a covalent system. First, the adapter cannot dissociate, which means there are no gaps during stochastic detection, a crucial advance for single-molecule exonuclease DNA sequencing where the continuous presence of a molecular adapter will be essential for reading individual nucleotides. Second, the ability to orient the adapter allows analytes to bind through only one of the two entrances to the betaCD cavity. We demonstrate that the covalently attached adapters can be used to alter the ion selectivity of the alphaHL pore, examine binding events at elevated temperatures, and detect analytes with prolonged dwell times.     

Single-molecule electrophoresis of beta-hairpin peptides by electrical recordings and Langevin dynamics simulations

We used single-channel electrical recordings and Langevin molecular dynamics simulations to explore the electrophoretic translocation of various beta-hairpin peptides across the staphylococcal alpha-hemolysin (alphaHL) protein pore at single-molecule resolution. The beta-hairpin peptides, which varied in their folding properties, corresponded to the C terminal residues of the B1 domain of protein G. The translocation time was strongly dependent on the electric force and was correlated with the folding features of the beta-hairpin peptides. Highly unfolded peptides entered the pore in an extended conformation, resulting in fast single-file translocation events. In contrast, the translocation of the folded beta-hairpin peptides occurred more slowly. In this case, the beta-hairpin peptides traversed the alphaHL pore in a misfolded or fully folded conformation. This study demonstrates that the interaction between a polypeptide and a beta-barrel protein pore is dependent on the folding features of the polypeptide.     

Electrostatic-gated transport in chemically modified glass nanopore electrodes

Electrostatic-gated transport in chemically modified glass nanopore electrodes with orifice radii as small as 15 nm is reported. A single conical-shaped nanopore in glass, with a approximately 1 microm radius Pt disk located at the pore base, is prepared by etching the exposed surface of a glass-sealed Pt nanodisk. The electrochemical response of the nanopore electrode corresponds to diffusion of redox-active species through the nanopore orifice to the Pt microdisk. Silanization of the exterior glass surface with Cl(Me)(2)Si(CH(2))(3)CN and the interior pore surface with EtO(Me)(2)Si(CH(2))(3)NH(2) introduces pH-dependent ion selectivity at the pore orifice, a consequence of the electrostatic interactions between the redox ions and protonated surface amines. Nanopore electrodes with very small pore orifice radii (< approximately 50 nm) display anion permselectively at pH < 4, as demonstrated by electrochemical measurement of transport through the pore orifice. Ion selective transport vanishes at pH > 6 or when the pore radius is significantly larger than the Debye screening length, consistent with the observed ion selectivity resulting from electrostatic interactions. The ability to introduce different surface functionalities to the interior and exterior surfaces of glass nanopores is demonstrated using fluorescence microscopy to monitor the localized covalent attachment of 5- (and 6)-carboxytetramethylrhodamine succinimidyl ester to interior pore surfaces previously silanized with EtO(Me)(2)Si(CH(2))(3)NH(2).     

Site-Specific Introduction of Bioorthogonal Handles to Nanopores by Genetic Code Expansion

Site-specific functionalization of natural amino acid-containing biological nanopores is pivotal in single molecule sensing. However, pore engineering methodologies are restricted to a limited choice and introduction of unnatural chemical components is extremely difficult. Herein we report the genetic code expansion (GCE) strategy to introduce unnatural amino acid (UAA) to an octameric Mycobacterium smegmatis porin A (MspA) nanopore. GCE allows for rapid and efficient introduction of bioorthogonal reactive site (i.e., azide) to the pore rim, and conjugation of single stranded DNA or lysozyme was demonstrated. The lysozyme-conjugated pore was further used for the discrimination of different oligosaccharides, demonstrating a sensing capacity that a bare MspA nanopore does not possess. GCE with bioorthogonal handles, which has never been previously applied in the preparation of nanopores, is a versatile strategy for pore engineering and may further expand the application scenarios of nanopores.     

Photoisomerization of an individual azobenzene molecule in water: an on-off switch triggered by light at a fixed wavelength

The alpha-hemolysin (alphaHL) pore was used as a nanoreactor for the direct observation of the reversible photoisomerization of individual tethered azobenzene molecules in an aqueous environment. alphaHL pores, PAZO, were used that had been derivatized within the lumen at a single cysteine residue with 4-((4-(2-chloroethanoamido)phenyl)diazenyl)benzenesulfonate. Trans-cis isomerizations were monitored at the single-molecule level by observing the modulation of the current passing through PAZO by electrical recording in planar bilayers. When PAZO was irradiated at 330 nm, continuous interconversion between the trans and cis states was observed. Either the trans or the cis state was maintained in the dark, depending upon which was present when the light source was shuttered. The cis state of PAZO was surprisingly stable in the dark, and no cis --> trans transitions were seen over a total observation period of more than 8 h. Therefore, based on our findings, it might be possible to make fast digital nanoscale switches operated by light of a fixed wavelength.     

Catalyzing the translocation of polypeptides through attractive interactions

Facilitated translocation of polypeptides through a protein pore is a ubiquitous and fundamental process in biology. Several translocation systems possess various well-defined binding sites within the pore lumen, but a clear mechanistic understanding of how the interaction of the polypeptides with the binding site alters the underlying kinetics is still missing. Here, we employed rational protein design and single-channel electrical recordings to obtain detailed kinetic signatures of polypeptide translocation through the staphylococcal alpha-hemolysin (alphaHL) transmembrane pore, a robust, tractable, and versatile beta-barrel protein. Acidic binding sites composed of rings of negatively charged aspartic acid residues, engineered at strategic positions within the beta barrel, produced dramatic changes in the functional properties of the alphaHL protein, facilitating the transport of cationic polypeptides from one side of the membrane to the other. When two electrostatic binding sites were introduced, at the entry and exit of the beta barrel, both the rate constants of association and dissociation increased substantially, diminishing the free energy barrier for translocation. By contrast, more hydrophobic polypeptides exhibited a considerable decrease in the rate constant of association to the pore lumen, having to overcome a greater energetic barrier because of the hydrophilic nature of the pore interior.     

Control of the Conductance of Engineered Protein Nanopores through Concerted Loop Motions

Protein nanopores have attracted much interest for nucleic acid sequencing, chemical sensing, and protein folding at the single molecule level. The outer membrane protein OmpG from E. coli stands out because it forms a nanopore from a single polypeptide chain. This property allows the separate engineering of each of the seven extracellular loops that control access to the pore. The longest of these loops, loop 6, has been recognized as the main gating loop that closes the pore at low pH values and opens it at high pH values. A method was devised to pin each of the loops to the embedding membrane and measure the single‐pore conductances of the resulting constructs. The electrophysiological and complementary NMR measurements show that the pinning of individual loops alters the structure and dynamics of neighboring and distant loops in a correlated fashion. Pinning loop 6 generates a constitutively open pore and patterns of concerted loop motions control access to the OmpG nanopore.     

纳米孔道单分子电化学测量的低噪音控温系统研究

纳米孔道检测技术是一种利用单个分子测量界面实现在单分子水平上测量DNA、RNA、蛋白、多肽等生物分子的高灵敏的单分子检测技术. 由于单个分子与纳米孔道的相互作用受热力学控制,亟需精准控制纳米孔道单分子分析的实验温度. 因此,本文研制了一种低噪音控温系统用于具有皮安级电流分辨的纳米孔道单分子实验,以实现精确调控测量时的环境温度. 该系统利用半导体制冷片的热电效应对检测池环境加热/制冷,通过对高精度热敏电阻进行电磁屏蔽以实现在温度反馈的同时避免噪音的引入. 利用比例-积分-微分算法进行控制,达到高精度快速控温的要求. 该系统控温精度为±1 °C,无额外噪音引入至超灵敏纳米孔道单分子测量,获得了25 °C到5 °C下Poly(dA)5与单个气单胞菌溶素（Aerolysin）分子界面间作用产生信号的差异,应用于研究单分子与纳米孔道相互作用的热力学行为.     

Advances in DNA Origami Nanopores: Fabrication,Characterization and Applications

In recent years, bio‐nanopore and solid‐state nanopore have been greatly improved for molecule bio‐sensing. Whereas, the development of this scientific field seems to have encountered a bottleneck due to their respective limitations. The small pore size of the former impedes the detection of large single molecule, and the latter is difficult to achieve similar accuracy and functional control. DNA origami plays a novel role to bring more opportunities for the development of nanopore technology since it is relatively easy to synthesize and modify. This review mainly focuses on introducing the DNA origami nanopore fabrication methods, characterization and application. Meanwhile, the challenges in the present DNA origami nanopore research are also discussed.     

Polymer capture by electro-osmotic flow of oppositely charged nanopores

The authors have addressed theoretically the hydrodynamic effect on the translocation of DNA through nanopores. They consider the cases of nanopore surface charge being opposite to the charge of the translocating polymer. The authors show that, because of the high electric field across the nanopore in DNA translocation experiments, electro-osmotic flow is able to create an absorbing region comparable to the size of the polymer around the nanopore. Within this capturing region, the velocity gradient of the fluid flow is high enough for the polymer to undergo coil-stretch transition. The stretched conformation reduces the entropic barrier of translocation. The diffusion limited translocation rate is found to be proportional to the applied voltage. In the authors' theory, many experimental variables (electric field, surface potential, pore radius, dielectric constant, temperature, and salt concentration) appear through a single universal parameter. They have made quantitative predictions on the size of the adsorption region near the pore for the polymer and on the rate of translocation.     

Resistive-pulse detection of multilamellar liposomes

The resistive-pulse method was used to monitor the pressure-driven translocation of multilamellar liposomes with radii between 190 and 450 nm through a single conical nanopore embedded in a glass membrane. Liposomes (0% and 5% 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (sodium salt) in 1,2-dilauroyl-sn-glycero-3-phosphocholine or 0%, 5%, and 9% 1,2-dipalmitoyl-sn-glycero-3-phospho(1'-rac-glycerol) (sodium salt) in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine) were prepared by extrusion through a polycarbonate membrane. Liposome translocation through a glass nanopore was studied as a function of nanopore size and the temperature relative to the lipid bilayer transition temperature, T(c). All translocation events through pores larger than the liposome, regardless of temperature, show translocation times between 30 and 300 μs and current pulse heights between 0.2% and 15% from the open pore baseline. However, liposomes at temperatures below the T(c) were captured at the pore orifice when translocation was attempted through pores of smaller dimensions, but squeezed through the same pores when the temperature was raised above T(c). The results provide insights into the deformation and translocation of individual liposomes through a porous material.     

Fluorescence microscopy of the pressure-dependent structure of lipid bilayers suspended across conical nanopores

Glass and fused-quartz nanopore membranes containing a single conically shaped pore are promising solid supports for lipid bilayer ion-channel recordings due to the high inherent stability of lipid bilayers suspended across the nanopore orifice, as well as the favorable electrical properties of glass and fused quartz. Fluorescence microscopy is used here to investigate the structure of the suspended lipid bilayer as a function of the pressure applied across a fused-quartz nanopore membrane. When a positive pressure is applied across the bilayer, from the nanopore interior relative to the exterior bulk solution, insertion or reconstitution of operative ion channels (e.g., α-hemolysin (α-HL) and gramicidin) in the bilayer is observed; conversely, reversing the direction of the applied pressure results in loss of all channel activity, although the bilayer remains intact. The dependence of the bilayer structure on pressure was explored by imaging the fluorescence intensity from Nile red dye doped into suspended 1,2-diphytanoyl-sn-glycero-3-phosphocholine bilayers, while simultaneously recording the activity of an α-HL channel. The fluorescence images suggest that a positive pressure results in compression of the bilayer leaflets and an increase in the bilayer curvature, making it suitable for ion-channel formation and activity. At negative pressure, the fluorescence images are consistent with separation of the lipid leaflets, resulting in the observed loss of the ion-channel activity. The fluorescence data indicate that the changes in the pressure-induced bilayer structure are reversible, consistent with the ability to repeatedly switch the ion-channel activity on and off by applying positive and negative pressures, respectively.     

AC conductance of transmembrane protein channels. The number of ionized residue mobile counterions at infinite dilution

Simultaneous measurements of the AC and DC conductances of alpha-hemolysin (alphaHL) ion channels and outer membrane protein F (OmpF) porins in dilute ionic solutions is described. AC conductance measurements were performed by applying a 10 mV rms AC voltage across a suspended planar bilayer of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine in the absence and presence of the protein and detecting the AC current response using phase-sensitive lock-in techniques. The conductances of individual alphaHL channels and OmpF porins were measured in symmetric KCl solutions containing between 5 and 1000 mM KCl. The AC and DC conductances of each protein were in agreement for all solution conditions, demonstrating the reliability of the AC method in single-channel recordings. Linear plots of conductance versus bulk KCl concentration for both proteins extrapolate to significant nonzero conductances (0.150 +/- 0.050 nS and 0.028 +/- 0.008 nS for OmpF and alphaHL, respectively) at infinite KCl dilution. The infinite dilution conductances are ascribed to mobile counterions of the ionizable residues within the protein lumens. A method of analyzing the plots of conductance vs KCl concentration is introduced that allows the determination of the concentration of mobile counterions associated with ionizable groups without knowledge of either the protein geometry or the ion mobilities. At neutral pH, an equivalent of 3 mobile counterions (K+ or Cl-) is estimated to contribute to the conductivity of the alphaHL channel.     

Oscillations in the capacitance of a nanopore containing an electrolyte due to pore width and nonzero size ions

Jiang, Jin, and Wu (Nano Lett., 11 (2011), pp. 5373-5377) have reported the results of a density functional theory (DFT) study of the capacitance of a nanopore containing an electrolyte consisting of charged hard spheres of equal diameter and charge. They find that the capacitance of the nanopore and electrolyte oscillates. The 'period' of the oscillations is of the order of the ionic diameter. Intuitively, the capacitance should tend to zero when the pore diameter is too small to accommodate the electrolyte ions and, thus, would contains no charge. A superficial glance at their table of contents figure might lead one to think that this does not occur in their calculation. However, they do not report results for exceedingly small pore diameters. In order to gain insight into their results, the Poisson-Boltzmann (PB) theory, which does not account for the diameter, is examined briefly. Unsurprisingly, the PB capacitance decreases monotonically to zero as the pore diameter decreases. The effect of a nonzero ion diameter is included in a semi-empirical manner by appealing to the results of the mean spherical approximation (MSA). The resulting capacitance oscillates and is qualitatively similar to the DFT results; it is zero at small pore diameter.     

Fabrication and functionalization of nanochannels by electron-beam-induced silicon oxide deposition

We report on the fabrication and electrical characterization of functionalized solid-state nanopores in low stress silicon nitride membranes. First, a pore of approximately 50 nm diameter was drilled using a focused ion beam technique, followed by the local deposition of silicon dioxide. A low-energy electron beam induced the decomposition of adsorbed tetraethyl orthosilicate resulting in site-selective functionalization of the nanopore by the formation of highly insulating silicon oxide. The deposition occurs monolayer by monolayer, which allows for control of the final diameter with subnanometer accuracy. Changes in the pore diameter could be monitored in real time by scanning electron microscopy. Recorded ion currents flowing through a single nanopore revealed asymmetry in the ion conduction properties with the sign of the applied potential. The low-frequency excess noise observed at negative voltage originated from stepwise conductance fluctuations of the open pore.     

Electrokinetic particle translocation through a nanopore containing a floating electrode

Electrokinetic particle translocation through a nanopore containing a floating electrode is investigated by solving a continuum model, composed of the coupled Poisson-Nernst-Planck (PNP) equations for the ionic mass transport and the modified Stokes equations for the flow field. Two effects due to the presence of the floating electrode, the induced-charge electroosmosis (ICEO) and the particle-floating electrode electrostatic interaction, could significantly affect the electrokinetic mobility of DNA nanoparticles. When the electrical double layers (EDLs) of the DNA nanoparticle and the floating electrode are not overlapped, the particle-floating electrode electrostatic interaction becomes negligible. As a result, the DNA nanoparticle could be trapped near the floating electrode arising from the induced-charge electroosmosis when the applied electric field is relatively high. The presence of the floating electrode attracts more ions inside the nanopore resulting in an increase in the ionic current flowing through the nanopore; however, it has a limited effect on the deviation of the current from its base current when the particle is far from the pore.     

Encapsulating a single G-quadruplex aptamer in a protein nanocavity

The alpha-hemolysin (alphaHL) protein pore has many applications in biotechnology. This article describes a single-molecule manipulation system that utilizes the nanocavity enclosed by this pore to noncovalently encapsulate a guest molecule. The guest is the thrombin-binding aptamer (TBA) that folds into the G-quadruplex in the presence of cations. Trapping the G-quadruplex in the nanocavity resulted in characteristic changes to the pore conductance that revealed important molecular processes, including spontaneous unfolding of the quartet structure and translocation of unfolded DNA in the pore. Through detection with Tag-TBA, we localized the G-quadruplex near the entry of the beta-barrel inside the nanocavity, where the molecule vibrates and rotates to different orientations. This guest-nanocavity supramolecular system has potential for helping to understand single-molecule folding and unfolding kinetics.