Single-channel Electrical Recordings Research Articles

Confirming Silent Translocation through Nanopores with Simultaneous Single-Molecule Fluorescence and Single-Channel Electrical Recordings.

Most of what is known concerning the luminal passage of materials through nanopores arises from electrical measurements. Whether nanopores are biological, solid-state, synthetic, hybrid, glass-capillary-based, or protein ion channels in cells and tissues, characteristic signatures embedded in the flow of ionic current are foundational to understanding functional behavior. In contrast, this work describes passage through a nanopore that occurs without producing an electrical signature. We refer to the phenomenon as "silent translocation." By definition, silent translocations are invisible to the standard tools of electrophysiology and fundamentally require a simultaneous ancillary measurement technique for positive identification. As a result, this phenomenon has been largely unexplored in the literature. Here, we report on a derivative of Cyanine 5 (sCy5a) that passes through the α-hemolysin (αHL) nanopore silently. Simultaneously acquired single-molecule fluorescence and single-channel electrical recordings from bilayers formed over a closed microcavity demonstrate that translocation does indeed take place, albeit infrequently. We report observations of silent translocation as a function of time, dye concentration, and nanopore population in the bilayer. Lastly, measurement of the translocation rate as a function of applied potential permits estimation of an effective energy barrier for transport through the pore as well as the effective charge on the dye, all in the absence of an information-containing electrical signature.

Synthetic α-Helical Nanopore Reactor for Chemical Sensing.

The use of nanopores for the single-molecule sensing of folded proteins and biomacromolecules has recently gained attention. Here, we introduce a simplified synthetic α-helical transmembrane pore, pPorA, as a nanoreactor and sensor that exhibits functional versatility comparable to that of engineered protein and DNA nanopores. The pore, built from the assembly of synthetic 40-amino-acid-long peptides, is designed to contain cysteine residues within the lumen and at the pore terminus for site-specific chemical modification probed using single-channel electrical recordings. The reaction of the pore with differently charged activated thiol reagents was studied, wherein positively charged reagents electrophoretically driven into the pore resulted in pore blocking in discrete steps upon covalent bond formation. The asymmetric blockage patterns resulting from cis and trans-side addition of reagents reveal the pore orientation in the lipid membrane. Furthermore, activated PEG thiols covalently blocked the pores over a longer duration in a charge-independent manner, establishing the large diameter and orientation of the formed pores. While the covalent binding of thiol reagents caused a drop in the pore conductance, cationic cyclic octasaccharides produced time-resolved translocation events, confirming the structural flexibility and tunability of the pores. The ability of the pore to accommodate large analytes and the considerable current amplitude variation following bond formation events are promising for developing platforms to resolve multistep chemical reactions at the single-molecule level for applications in synthetic nanobiotechnology.

Constructing ion channels from water-soluble α-helical barrels.

The design of peptides that assemble in membranes to form functional ion channels is challenging. Specifically, hydrophobic interactions must be designed between the peptides and at the peptide-lipid interfaces simultaneously. Here, we take a multi-step approach towards this problem. First, we use rational de novo design to generate water-soluble α-helical barrels with polar interiors, and confirm their structures using high-resolution X-ray crystallography. These α-helical barrels have water-filled lumens like those of transmembrane channels. Then, we modify the sequences to facilitate their insertion into lipid bilayers. Single-channel electrical recordings and fluorescent imaging of the peptides in membranes show monodisperse, cation-selective channels of unitary conductance. Surprisingly, however, an X-ray structure solved from lipidic cubic phase for one peptide reveals an alternative state with tightly packed helices and a constricted channel. To reconcile these observations, we perform computational analyses to compare the properties of possible different states of the peptide.

Transmembrane Epitope Delivery by Passive Protein Threading through the Pores of the OmpF Porin Trimer.

Trimeric porins in the outer membrane (OM) of Gram-negative bacteria are the conduits by which nutrients and antibiotics diffuse passively into cells. The narrow gateways that porins form in the OM are also exploited by bacteriocins to translocate into cells by a poorly understood process. Here, using single-channel electrical recording in planar lipid bilayers in conjunction with protein engineering, we explicate the mechanism by which the intrinsically unstructured N-terminal translocation domain (IUTD) of the endonuclease bacteriocin ColE9 is imported passively across the Escherichia coli OM through OmpF. We show that the import is dominated by weak interactions of OmpF pores with binding epitopes within the IUTD that are orientationally biased and result in the threading of over 60 amino acids through 2 subunits of OmpF. Single-molecule kinetic analysis demonstrates that the IUTD enters from the extracellular side of OmpF and translocates to the periplasm where the polypeptide chain does an about turn in order to enter a neighboring subunit, only for some of these molecules to pop out of this second subunit before finally re-entering to form a stable complex. These intimately linked transport/binding processes generate an essentially irreversible, hook-like assembly that constrains an import activating peptide epitope between two subunits of the OmpF trimer.

Autonomously Assembled Synthetic Transmembrane Peptide Pore.

The porinACj is an α-helical porin that spans the mycolic acid outer membrane of Gram-positive mycolate, Corynebacterium jeikeium. Here, we report that a 40-aminoacid, synthetic peptide, pPorA corresponding to porin PorACj, inserts into the lipid bilayers and forms well-defined pores. By electrical recordings, we measured the single-channel properties that revealed the autonomous assembly of large conductance ion-selective synthetic pores. Further, we characterized the functional properties by blocking the peptide pores by cyclodextrins of different charge and symmetry. We deduced the subunit stoichiometry and putative structure of the pore by site-specific chemical modification in single-channel electrical recordings and gel electrophoresis. On the basis of these findings, we suggest that this is a large functional uniform transmembrane pore built entirely from short synthetic α-helical peptides. Accordingly, we propose a model demonstrating structural assembly of large α-helix-based peptide pores for understanding the action of antimicrobial peptides and for the design of pores with applications in biotechnology.

A new class of hybrid secretion system is employed in Pseudomonas amyloid biogenesis

Gram-negative bacteria possess specialised biogenesis machineries that facilitate the export of amyloid subunits for construction of a biofilm matrix. The secretion of bacterial functional amyloid requires a bespoke outer-membrane protein channel through which unfolded amyloid substrates are translocated. Here, we combine X-ray crystallography, native mass spectrometry, single-channel electrical recording, molecular simulations and circular dichroism measurements to provide high-resolution structural insight into the functional amyloid transporter from Pseudomonas, FapF. FapF forms a trimer of gated β-barrel channels in which opening is regulated by a helical plug connected to an extended coil-coiled platform spanning the bacterial periplasm. Although FapF represents a unique type of secretion system, it shares mechanistic features with a diverse range of peptide translocation systems. Our findings highlight alternative strategies for handling and export of amyloid protein sequences.

Imaging the dynamics of individual electropores.

Electroporation is a widely used technique to permeabilize cell membranes. Despite its prevalence, our understanding of the mechanism of voltage-mediated pore formation is incomplete; methods capable of visualizing the time-dependent behavior of individual electropores would help improve our understanding of this process. Here, using optical single-channel recording, we track multiple isolated electropores in real time in planar droplet interface bilayers. We observe individual, mobile defects that fluctuate in size, exhibiting a range of dynamic behaviors. We observe fast (25 s(-1)) and slow (2 s(-1)) components in the gating of small electropores, with no apparent dependence on the applied potential. Furthermore, we find that electropores form preferentially in the liquid disordered phase. Our observations are in general supportive of the hydrophilic toroidal pore model of electroporation, but also reveal additional complexity in the interactions, dynamics, and energetics of electropores.

Channel-forming bacterial toxins in biosensing and macromolecule delivery.

To intoxicate cells, pore-forming bacterial toxins are evolved to allow for the transmembrane traffic of different substrates, ranging from small inorganic ions to cell-specific polypeptides. Recent developments in single-channel electrical recordings, X-ray crystallography, protein engineering, and computational methods have generated a large body of knowledge about the basic principles of channel-mediated molecular transport. These discoveries provide a robust framework for expansion of the described principles and methods toward use of biological nanopores in the growing field of nanobiotechnology. This article, written for a special volume on “Intracellular Traffic and Transport of Bacterial Protein Toxins”, reviews the current state of applications of pore-forming bacterial toxins in small- and macromolecule-sensing, targeted cancer therapy, and drug delivery. We discuss the electrophysiological studies that explore molecular details of channel-facilitated protein and polymer transport across cellular membranes using both natural and foreign substrates. The review focuses on the structurally and functionally different bacterial toxins: gramicidin A of Bacillus brevis, α-hemolysin of Staphylococcus aureus, and binary toxin of Bacillus anthracis, which have found their “second life” in a variety of developing medical and technological applications.

Shaped Apertures in Photoresist Films Enhance the Lifetime and Mechanical Stability of Suspended Lipid Bilayers

Planar lipid bilayers suspended in apertures provide a controlled environment for ion channel studies. However, short lifetimes and poor mechanical stability of suspended bilayers limit the experimental throughput of bilayer electrophysiology experiments. Although bilayers are more stable in smaller apertures, ion channel incorporation through vesicle fusion with the suspended bilayer becomes increasingly difficult. In an alternative bilayer stabilization approach, we have developed shaped apertures in SU8 photoresist that have tapered sidewalls and a minimum diameter between 60 and 100 μm. Bilayers formed at the thin tip of these shaped apertures, either with the painting or the folding method, display drastically increased lifetimes, typically >20 h, and mechanical stability, being able to withstand extensive perturbation of the buffer solution. Single-channel electrical recordings of the peptide alamethicin and of the proteoliposome-delivered potassium channel KcsA demonstrate channel conductance with low noise, made possible by the small capacitance of the 50 μm thick SU8 septum, which is only thinned around the aperture, and unimpeded proteoliposome fusion, enabled by the large aperture diameter. We anticipate that these shaped apertures with micrometer edge thickness can substantially enhance the throughput of channel characterization by bilayer lipid membrane electrophysiology, especially in combination with automated parallel bilayer platforms.

An engineered dimeric protein pore that spans adjacent lipid bilayers

The bottom-up construction of artificial tissues is an underexplored area of synthetic biology. An important challenge is communication between constituent compartments of the engineered tissue and between the engineered tissue and additional compartments, including extracellular fluids, further engineered tissue and living cells. Here we present a dimeric transmembrane pore that can span two adjacent lipid bilayers and thereby allow aqueous compartments to communicate. Two heptameric staphylococcal α-hemolysin (αHL) pores were covalently linked in an aligned cap-to-cap orientation. The structure of the dimer, (α7)2, was confirmed by biochemical analysis, transmission electron microscopy (TEM) and single-channel electrical recording. We show that one of two β barrels of (α7)2 can insert into the lipid bilayer of a small unilamellar vesicle, while the other spans a planar lipid bilayer. (α7)2 pores spanning two bilayers were also observed by TEM.

Gangliosides Mediate a Two-Step Mechanism of Membrane Disruption by Beta-Amyloid: Initial Pore Formation Followed by Membrane Fragmentation

The mechanism of amyloid-β (Aβ) toxicity has long been a topic of intense debate. One prevailing hypothesis states that cytotoxicity arises through Aβ interacting with and disrupting cell membranes. Here, we demonstrate that membrane disruption by Aβ occurs via a two-step process, with the initial formation of ion-selective pores followed by nonspecific fragmentation of the lipid membrane during amyloid fiber formation. Immediately after the addition of freshly dissolved Aβ(1-40), defects form on the membrane that share many of the properties of the Aβ ion-channel-like pores originally reported from single-channel electrical recording, such as cation selectivity and the ability to be blockaded by zinc. By contrast, the fiber-dependent step of membrane disruption by Aβ(1-40) involves a detergent-like mechanism characterized by fragmentation of the membrane that is neither cation ion selective nor blockaded by Zn2+. Moreover, we observed that the presence of ganglioside enhances both the initial pore formation and the fiber-dependent membrane fragmentation process. In addition, using a combinational technique of solid-state and solution-state NMR spectroscopy, we show that Aβ(1-40) fibrils are capable of binding to ganglioside-containing membranes, potentially providing a beacon for the mechanism of nonspecific membrane fragmentation. This fiber-dependent membrane fragmentation can only be observed in the presence gangliosides, whereas pore formation by freshly dissolved Aβ(1-40) is weakly observed in their absence. These results provide insights into the toxicity of Aβ and may aid in the design of specific compounds to alleviate the neurodegeneration of Alzheimer's disease.Sciacca, M. F., Kotler, S. A., Brender, J. R., Chen, J., Lee, D. K., and Ramamoorthy, A. (2012) Biophys J 103, 702-710

Biomolecular Detection with Engineered Robust Nanopores

Nanopores have become indispensable, powerful tools for bimolecular detections and molecular manipulations at single-molecule level, and they have been realized as an alternative to current analytical devices in biomedical molecular diagnosis. With a limited number of protein nanopores available, designing robust, functionally-intact protein scaffolds under various detection conditions is a daunting challenge in developing protein nanopore-based technologies. We used ferric hydroxamate uptake component A (FhuA), a bacterial β-barrel membrane protein as a template for protein engineering to produce an exceptionally-functional nanopore for sampling biomolecular events in harsh environments.FhuA has a luminal cross-section of ∼3.1×4.4 nm and is plugged by a globular N-terminal cork domain (C). Single-channel electrical recordings with extensive protein redesign of FhuA resulted in identifying four long extracellular loops (4L) that partially block the lumen upon the removal of the cork. The newly engineered protein, FhuA ΔC/Δ4L, was the result of a removal of almost 33% of the total number of amino acids of the wild-type FhuA (WT-FhuA) protein. The crown achievement in this work was combining direct genetic engineering with a refolding approach to produce this unusually-stable protein nanopore. Critical to its future nanotechnological applications, FhuA ΔC/Δ4L was functional under structure-altering conditions, including low ion concentration and highly acidic aqueous phase.To tailor the FhuAΔC/Δ4L protein to the use in nanopore-based detection devices, we show that the FhuA-based nanopores function as stochastic biosensing elements. For example, we monitored the proteolytic activity of an enzyme at highly acidic pH and we were able to determine the kinetics of protein-DNA aptamer interactions at physiological salt concentration. These two assays have not been demonstrated with the existing nanopores.This work is funded in part by grants from the US National Science Foundation (DMR-1006332, L.M.) and the National Institutes of Health (R01 GM088403, L.M.).

Two-Step Mechanism of Membrane Disruption by Aβ through Membrane Fragmentation and Pore Formation

Disruption of cell membranes by Aβ is believed to be one of the key components of Aβ toxicity. However, the mechanism by which this occurs is not fully understood. Here, we demonstrate that membrane disruption by Aβ occurs by a two-step process, with the initial formation of ion-selective pores followed by nonspecific fragmentation of the lipid membrane during amyloid fiber formation. Immediately after the addition of freshly dissolved Aβ1–40, defects form on the membrane that share many of the properties of Aβ channels originally reported from single-channel electrical recording, such as cation selectivity and the ability to be blockaded by zinc. By contrast, subsequent amyloid fiber formation on the surface of the membrane fragments the membrane in a way that is not cation selective and cannot be stopped by zinc ions. Moreover, we observed that the presence of ganglioside enhances both the initial pore formation and the fiber-dependent membrane fragmentation process. Whereas pore formation by freshly dissolved Aβ1–40 is weakly observed in the absence of gangliosides, fiber-dependent membrane fragmentation can only be observed in their presence. These results provide insights into the toxicity of Aβ and may aid in the design of specific compounds to alleviate the neurodegeneration of Alzheimer’s disease.

Rationale Membrane Protein Design of a Beta-Barrel

Bacterial outer membrane proteins have robust beta-barrel structures. Therefore, they are targeted for engineering biological nanopores as stochastic biosensing elements. We used membrane protein engineering and single-channel electrical recordings to explore the ferric hydroxamate uptake component A (FhuA) as a target for redesigning membrane proteins. FhuA is a monomeric 22-stranded beta-barrel protein from the outer membrane of Escherichia coli. FhuA has a lumen with cross section of 3.1 X 4.4 nm that is filled by a globular N-terminal cork domain. We investigated various redesigned FhuA proteins, which had either single, double, or multiple deletions of the large extracellular loops and the cork domain. Analysis of the electrical signatures of these initial exploratory redesigned FhuA proteins led to the identification of four large extracellular loops that partially occlude the lumen when the cork domain is removed. Accordingly, we removed the cork along with the extracellular loops, FhuA delta C/delta 4L, resulting in the deletion of almost one third of the total number of amino acids of the wild-type FhuA protein. Remarkably, the newly redesigned protein forms an open pore in planar lipid bilayers, with a measured unitary conductance of ∼4.8 nanosiemens in 1 M KCl at pH 7.4, a value that has not been recorded previously with other engineered FhuA protein channels. We show several advantages and prospects of using such an engineered outer membrane protein in fundamental studies of membrane protein folding and design, and the mechanisms of ion conductance and gating. Further, FhuA delta C/delta 4L can be a platform for customized engineering in applicative areas of single-molecule sensing and analysis of proteins, nucleic acids and their ensembles.

Dynamic and Reversible Control of 2D Membrane Protein Concentration in a Droplet Interface Bilayer

We form an artificial lipid bilayer between a nanolitre aqueous droplet and a supporting hydrogel immersed in an oil/lipid solution. Manipulation of the axial position of the droplet relative to the hydrogel controls the size of the bilayer formed at the interface; this enables the surface density of integral membrane proteins to be controlled. We are able to modulate the surface density of the β-barrel pore-forming toxin α-hemolysin over a range of 4 orders of magnitude within a time frame of a few seconds. The concentration changes are fully reversible. Membrane protein function and diffusion are unaltered, as measured by single molecule microscopy and single channel electrical recording.

Analysis of gating transitions among the three major open states of the OpdK channel.

OpdK is an outer membrane protein of the pathogenic bacterium Pseudomonas aeruginosa. The recent crystal structure of this protein revealed a monomeric, 18-stranded β-barrel with a kidney-shaped pore, whose constriction features a diameter of 8 Å. Using systematic single-channel electrical recordings of this protein pore reconstituted into planar lipid bilayers under a broad range of ion concentrations, we were able to probe its discrete gating kinetics involving three major and functionally distinct conformations, in which a dominant open substate O(2) is accompanied by less thermodynamically stable substates O(1) and O(3). Single-channel electrical data enabled us to determine the alterations in the energetics and kinetics of the OpdK protein when experimental conditions were changed. In the future, such a semiquantitative analysis might provide a better understanding on the dynamics of current fluctuations of other β-barrel membrane protein channels.

Redesign of a Plugged β-Barrel Membrane Protein

The redesign of biological nanopores is focused on bacterial outer membrane proteins and pore-forming toxins, because their robust β-barrel structure makes them the best choice for developing stochastic biosensing elements. Using membrane protein engineering and single-channel electrical recordings, we explored the ferric hydroxamate uptake component A (FhuA), a monomeric 22-stranded β-barrel protein from the outer membrane of Escherichia coli. FhuA has a luminal cross-section of 3.1 × 4.4 nm and is filled by a globular N-terminal cork domain. Various redesigned FhuA proteins were investigated, including single, double, and multiple deletions of the large extracellular loops and the cork domain. We identified four large extracellular loops that partially occlude the lumen when the cork domain is removed. The newly engineered protein, FhuAΔC/Δ4L, was the result of a removal of almost one-third of the total number of amino acids of the wild-type FhuA (WT-FhuA) protein. This extensive protein engineering encompassed the entire cork domain and four extracellular loops. Remarkably, FhuAΔC/Δ4L forms a functional open pore in planar lipid bilayers, with a measured unitary conductance of ∼4.8 nanosiemens, which is much greater than the values recorded previously with other engineered FhuA protein channels. There are numerous advantages and prospects of using such an engineered outer membrane protein not only in fundamental studies of membrane protein folding and design, and the mechanisms of ion conductance and gating, but also in more applicative areas of stochastic single-molecule sensing of proteins and nucleic acids.

Reversing the Charges within the Lumen of Alpha-Hemolysin Protein Pore Affects its Robustness

Staphylococcal alpha-hemolysin (αHL), a heptameric transmembrane protein of known high-resolution crystal structure, has been targeted for engineering for many biotechnological applications due to its high stability in planar lipid bilayers. Using single-channel electrical recordings and standard protein engineering, we explored how reversed charges within the interior of the beta-barrel part of the pore alter the protein robustness. We replaced the lysines with aspartic acids. Reversing charges within the pore constriction, the structurally stiff region of the beta barrel, reduced the conductance of the pore, but produced current fluctuation-free channel behavior. In contrast, reversing charges at the structurally flexible glycine-rich turn of the beta barrel, increased the conductance of the pore and produced gating activity in the form of large-amplitude and frequent current fluctuations. Remarkably, reversing charges within both regions caused a large-amplitude permanent current blockade of the beta barrel. Our results suggest that these distant charge reversals are energetically coupled.

Tuning the Cavity of Cyclodextrins: Altered Sugar Adaptors in Protein Pores

Cyclodextrins (CDs) have been widely used in host-guest molecular recognition because of their chiral and hydrophobic cavities. For example, β-cyclodextrin (βCD) lodged as a molecular adaptor in protein pores such as α-hemolysin (αHL) is used for stochastic sensing. Here, we have tuned the cavity and overall size of βCD by replacing a single oxygen atom in its ring skeleton by a disulfide unit in two different configurations to both expand our ability to detect analytes and understand the interactions of βCD with protein pores. The three-dimensional structures of the two stereoisomeric CDs have been determined by the combined application of NMR spectroscopy and molecular simulation and show distorted conformations as compared to natural βCD. The interactions of these synthetic βCD analogues with mutant αHL protein pores and guest molecules were studied by single-channel electrical recording. The dissociation rate constants for both disulfide CDs from the mutant pores show ∼1000-fold increase as compared to those of unaltered βCD, but are ∼10-fold lower than the dissociation rate constants for βCD from wild-type αHL. Both of the skeleton-modified CDs show altered selectivity toward guest molecules. Our approach expands the breadth in sensitivity and diversity of sensing with protein pores and suggests structural parameters useful for CD design, particularly in the creation of asymmetric cavities.

Interactions of Mitochondrial Presequence Peptides with the Mitochondrial Outer Membrane Preprotein Translocase TOM

TOM protein-conducting channels serve as the main entry sites into mitochondria for virtually all mitochondrial proteins. When incorporated into lipid bilayers, they form large, relatively nonspecific ion channels that are blocked by peptides derived from mitochondrial precursor proteins. Using single-channel electrical recordings, we analyzed the interactions of mitochondrial presequence peptides with single TOM pores. The largest conductance state of the translocon represents the likely protein-conducting conformation of the channel. The frequency (but not the duration) of the polypeptide-induced blockage is strongly modulated by the substrate concentration. Structural differences between substrates are reflected in characteristic blockage frequencies and duration of blockage. To our knowledge, this study provides first quantitative data regarding the kinetics of polypeptide interaction with the mitochondrial TOM machinery.