Cell-free protein synthesis (CFPS) has emerged as a powerful tool for the rapid synthesis and analysis of various structurally and functionally distinct proteins. These include ‘difficult-to-express’ membrane proteins such as large multipass ion channel receptors. Owing to their membrane localization, eukaryotic CFPS supplemented with endoplasmic reticulum (ER)-derived microsomal vesicles has proven to be an efficient system for the synthesis of functional membrane proteins. Here we demonstrate the applicability of the eukaryotic cell-free systems based on lysates from the mammalian Chinese Hamster Ovary (CHO) and insect Spodoptera frugiperda (Sf21) cells. We demonstrate the efficiency of the systems in the de novo cell-free synthesis of the human cardiac ion channels: ether-a-go-go potassium channel (hERG) KV11.1 and the voltage-gated sodium channel hNaV1.5.
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Nanopores are currently utilized as powerful tools for single-molecule protein sensing. The reporting signal typically requires protein analytes to enter the nanopore interior, yet a class of these sensors has emerged that allows targeted detection free in solution. This tactic eliminates the spatial limitation of nanopore confinement. However, probing proteins outside the nanopore implies numerous challenges associated with transducing the physical interactions in the aqueous phase into a reliable electrical signature. Hence, it necessitates extensive engineering and tedious optimization routes. These obstacles have prevented the widespread adoption of these sensors. Here, we provide an experimental strategy by developing and validating single-polypeptide-chain nanopores amenable to single-molecule and bulk-phase protein detection approaches. We utilize protein engineering, as well as nanopore and nanodisc technologies, to create nanopore sensors that can be integrated with an optical platform in addition to traditional electrical recordings. Using the optical modality over an ensemble of detectors accelerates these sensors’ optimization process for a specific task. It also provides insights into how the construction of these single-molecule nanopore sensors influences their performance. These outcomes form a basis for evaluating engineered nanopores beyond the fundamental limits of the resistive-pulse technique.
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The standard model of pore formation was introduced more than fifty years ago, and it has been since, despite some refinements, the cornerstone for interpreting experiments related to pores in membranes. A central prediction of the model concerning pore opening under an electric field is that the activation barrier for pore formation is lowered proportionally to the square of the electric potential. However, this has only been scarcely and inconclusively confronted to experiments. In this paper, we study the electropermeability of model lipid membranes composed of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) containing different fractions of POPC-OOH, the hydroperoxidized form of POPC, in the range 0 to 100 mol %. By measuring ion currents across a 50-μm-diameter black lipid membrane (BLM) with picoampere and millisecond resolution, we detect hydroperoxidation-induced changes to the intrinsic bilayer electropermeability and to the probability of opening angstrom-size or larger pores. Our results over the full range of lipid compositions show that the energy barrier to pore formation is lowered linearly by the absolute value of the electric field, in contradiction with the predictions of the standard model.
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Here, we demonstrate the utility of native membrane derived vesicles (nMVs) as tools for expeditious electrophysiological analysis of membrane proteins. We used a cell-free (CF) and a cell-based (CB) approach for preparing protein-enriched nMVs. We utilized the Chinese Hamster Ovary (CHO) lysate-based cell-free protein synthesis (CFPS) system to enrich ER-derived microsomes in the lysate with the primary human cardiac voltage-gated sodium channel 1.5 (hNaV1.5; SCN5A) in 3 h. Subsequently, CB-nMVs were isolated from fractions of nitrogen-cavitated CHO cells overexpressing the hNaV1.5. In an integrative approach, nMVs were micro-transplanted into Xenopus laevis oocytes. CB-nMVs expressed native lidocaine-sensitive hNaV1.5 currents within 24 h; CF-nMVs did not elicit any response. Both the CB- and CF-nMV preparations evoked single-channel activity on the planar lipid bilayer while retaining sensitivity to lidocaine application. Our findings suggest a high usability of the quick-synthesis CF-nMVs and maintenance-free CB-nMVs as ready-to-use tools for in-vitro analysis of electrogenic membrane proteins and large, voltage-gated ion channels.
Artificial lipid bilayers represent the gold standard for the investigation of membrane spanning species like purified ion channels or membrane-active species in general like toxins. However, the convenient and reproducible preparation of these model bilayers as well as the delicate successive handling for data acquisition still remain an obstacle for the trouble-free introduction of this technique. We here present Nanion’s Orbit systems that are explicitly designed to meet the special requirements of experiments on artificial bilayers. Use of Ionera’s MECA (micro electrode cavity array) chip technology combined with state of the art low noise amplifiers (Elements S.R.L.) enables the fully parallel low-noise recording of four or even 16 separate lipid bilayers at bandwidths up to 100 kHz. The systems have already been validated with targets as diverse as ligand and voltage gated ion channels, porins and origami DNA constructs, antimicrobial peptides or membrane active toxins. The optional temperature control for the Orbit mini furthermore allows for experiments on temperature sensitive species such as TRP channels or for experiments at physiological temperatures whereas the fully automated bilayer generation on the Orbit e16 further improves the system’s usability
Portable DNA sequencing and biosensing can advance research, bedside-diagnostics, and homeland security. I describe how label-free sensing is achieved with atom-scale designed membrane nanopores. In this strategy, nanopores act as electronic sensors that detect when individual molecules pass the pores’ nanoscale hole. The temporary blockages cause changes in ionic pore current. The approach has helped pioneer portable DNA sequencing with protein pores(1) to discriminate individual bases. More recently, synthetic pores have been built by folding DNA strands into defined channels(2). The DNA nanopores are relevant as they overcome the narrow size range of protein pores and thereby accommodate folded protein analytes. The DNA nanostructures are also easier to rationally design than proteins(3) and thereby enable new applications, also in synthetic biology(4).
(1) Nature 2014 516 250;
(2) Nat. Nanotechnol. 2016 11 152;
(3) Nat. Nanotechnol. 2017 12 619;
(4) Science 2016 352 890; Nat. Chem. 2017 9 611
This webinar covers the use of the lipid bilayer platforms from Nanion: the Orbit16 and the Orbit mini for characterization of membrane proteins like ion channels, bacterial porins and biological nanopores. Both bilayer systems support high quality low noise recordings, but differ in throughput capabilities and experimental features. The Orbit16, introduced in 2012 is a device for efficient formation of 16 lipid bilayers simultaneously, allowing for parallel bilayer-reconstitution of ion channels and nanopores
Lipid bilayer arrays are automatically formed by remotely actuated painting. Orbit 16 has proven to be a versatile device for research and scientific applications which is reflected by numerous publication in high rank journals such as Science, Nature, Nano Letters, ACS Nano etc. The Orbit mini, introduced in 2015, is a smaller system, allowing low noise, high bandwidth recordings from 4 bilayers simultaneously. Orbit mini supports temperature-controlled experiments, allowing both active cooling and heating, a benefit when investigating thermo-sensitive channels such as TRPA1 or TRPV1, which will be shown in this webinar. Micro Electrode Cavity Array chips (MECA chips, Ionera Technologies GmbH, Freiburg) are the core of both devices. The MECA chips have successfully been validated with a wide variety of different ion channels and pores including KcsA, gramicidin, α-hemolysin, Kv- and Nav-channels etc. In this webinar the basics of the MECA technology will be explained and its applications will be shown in detail. Join this webinar and see how these outstanding platforms can add up to your work: Orbit 16 and Orbit mini!
DNA nanopores are bio-inspired nanostructures that control molecular transport across lipid bilayer membranes. Researchers can readily engineer the structure and function of DNA nanopores to synergistically combine the strengths of DNA nanotechnology and nanopores. The pores can be harnessed in a wide range of areas, including biosensing, single-molecule chemistry, and single-molecule biophysics, as well as in cell biology and synthetic biology. Here, we provide a protocol for the rational design of nanobarrel-like DNA pores and larger DNA origami nanopores for targeted applications. We discuss strategies for the pores’ chemical modification with lipid anchors to enable them to be inserted into membranes such as small unilamellar vesicles (SUVs) and planar lipid bilayers. The procedure covers the self-assembly of DNA nanopores via thermal annealing, their characterization using gel electrophoresis, purification, and direct visualization with transmission electron microscopy and atomic force microscopy. We also describe a gel assay to determine pore–membrane binding and discuss how to use single-channel current recordings and dye flux assays to confirm transport through the pores. We expect this protocol to take approximately 1 week to complete for DNA nanobarrel pores and 2–3 weeks for DNA origami pores.
Chapter 4: Electrophysiology on Channel-Forming Proteins in Artificial Lipid Bilayers: Next-Generation Instrumentation for Multiple Recordings in Parallel Abstract: Artificial lipid bilayers have been used for several decades to study channel-forming pores and ion channels in membranes. Until recently, the classical two-chamber setups have been primarily used for studying the biophysical properties of pore forming proteins. Within the last 10 years, instruments for automated lipid bilayer measurements have been developed and are now commercially available. This chapter focuses on protein purification and reconstitution of channel-forming proteins into lipid bilayers using a classic setup and on the commercially available systems, the Orbit mini and Orbit 16.
Nanopores are key in portable sequencing and research given their ability to transport elongated DNA or small bioactive molecules through narrow transmembrane channels. Transport of folded proteins could lead to similar scientific and technological benefits. Yet this has not been realised due to the shortage of wide and structurally defined natural pores. Here we report that a synthetic nanopore designed via DNA nanotechnology can accommodate folded proteins. Transport of fluorescent proteins through single pores is kinetically analysed using massively parallel optical readout with transparent silicon-on-insulator CaVity chips vs. electrical recordings to reveal an at least 20-fold higher speed for the electrically driven movement. Pores nevertheless allow a high diffusive flux of more than 66 molecules per second that can also be directed beyond equillibria. The pores may be exploited to sense diagnostically relevant proteins with portable analysis technology, to create molecular gates for drug delivery, or to build synthetic cells.
Membrane-spanning nanopores from folded DNA are a recent example of biomimetic man-made nanostructures that can open up applications in biosensing, drug delivery, and nanofluidics. In this report, we generate a DNA nanopore based on the archetypal six-helix-bundle architecture and systematically characterize it via single-channel current recordings to address several fundamental scientific questions in this emerging field. We establish that the DNA pores exhibit two voltage-dependent conductance states. Low transmembrane voltages favor a stable high-conductance level, which corresponds to an unobstructed DNA pore. The expected inner width of the open channel is confirmed by measuring the conductance change as a function of poly(ethylene glycol) (PEG) size, whereby smaller PEGs are assumed to enter the pore. PEG sizing also clarifies that the main ion-conducting path runs through the membrane-spanning channel lumen as opposed to any proposed gap between the outer pore wall and the lipid bilayer. At higher voltages, the channel shows a main low-conductance state probably caused by electric-field-induced changes of the DNA pore in its conformation or orientation. This voltage-dependent switching between the open and closed states is observed with planar lipid bilayers as well as bilayers mounted on glass nanopipettes. These findings settle a discrepancy between two previously published conductances. By systematically exploring a large space of parameters and answering key questions, our report supports the development of DNA nanopores for nanobiotechnology.
Electrophysiological studies of the interaction of polymers with pores formed by bacterial toxins provide a window on single molecule interaction with proteins in real time, report on the behavior of macromolecules in confinement, and 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.
Cell-free protein synthesis (CFPS) based on eukaryotic Sf21 lysate is gaining interest among researchers due to its ability to handle the synthesis of complex human membrane proteins (MPs). Additionally Sf21 cell-free systems contain endogenous microsomal vesicles originally derived from the endoplasmic reticulum (ER). After CFPS, MPs will be translocated into the microsomal vesicles membranes present in the lysates. Thus microsomal membranes offer a natural environment for de novo synthesized MPs. Despite the advantage of synthesizing complex MPs with post translational modifications directly into the microsomal membranes without any additional solubilization supplements, batch based Sf21 cell-free synthesis suffers from low yields. The bottleneck for MPs in particular after the synthesis and incorporation into the microsomal membranes is to analyze their functionality. Apart from low yields of the synthesized MPs with batch based cell-free synthesis, the challenges arise in the form of cytoskeleton elements and peripheral endogenous proteins surrounding the microsomes which may impede the functional analysis of the synthesized proteins. So careful sample processing after the synthesis is particularly important for developing the appropriate functional assays. Here we demonstrate how MPs (native and batch synthesized) from ER derived microsomes can be processed for functional analysis by electrophysiology and radioactive uptake assay methods. Treatment of the microsomal membranes either with a sucrose washing step in the case of human serotonin transporter (hSERT) and sarco/endoplasmic reticulum Ca2+/ATPase (SERCA) pump or with mild detergents followed by the preparation of proteoliposomes in the case of the human voltage dependent anionic channel (hVDAC1) helps to analyze the functional properties of MPs.
In general, the method of choice to characterize the conductance properties of channel-forming bacterial porins is electrophysiology. Here, the classical method is to reconstitute single porins into planar lipid bilayers to derive functional information from the observed channel conductance. In addition to an estimated pore size, ion selectivity or transport properties in general are of importance. For the latter, measuring the ion current fluctuation can provide some information about the mode of transport of charged molecules penetrating the proteins. For instance, increasing the external voltage modifies the residence time in the channel: charged molecules with the ability to permeate through channels will travel faster whereas non-permeating molecules get pushed to the constriction zone with enhanced residence time. Here, we are interested in the ability of antibiotics to permeate channels and compare different techniques to reveal fast events.
Controlled transport of biomolecules across lipid bilayer membranes is of profound significance in biological processes. In cells, cargo exchange is mediated by dedicated channels that respond to triggers, undergo a nanomechanical change to reversibly open, and thus regulate cargo flux. Replicating these processes with simple yet programmable chemical means is of fundamental scientific interest. Artificial systems that go beyond nature’s remit in transport control and cargo are also of considerable interest for biotechnological applications but challenging to build. Here, we describe a synthetic channel that allows precisely timed, stimulus-controlled transport of folded and functional proteins across bilayer membranes. The channel is made via DNA nanotechnology design principles and features a 416 nm2 opening cross-section and a nanomechanical lid which can be controllably closed and re-opened via a lock-and-key mechanism. We envision that the functional DNA device may be used in highly sensitive biosensing, drug delivery of proteins, and the creation of artificial cell networks.
Membrane nanopores are key for molecular transport in biology, portable DNA sequencing, label-free single-molecule analysis and nanomedicine. Transport traditionally relies on barrel-like channels of a few nanometres width, but there is considerable scientific and technological interest for much wider structures of tunable shape. Yet, these nanopores do not exist in nature and are challenging to build using existing de novo routes for proteins. Here, we show that rational design with DNA can drastically expand the structural and functional range of membrane nanopores. Our design strategy bundles DNA duplexes into pore subunits that modularly arrange to form tunable pore shapes and lumen widths of up to tens of nanometres. Functional units for recognition or signalling can be optionally attached. By dialling in essential parameters, we demonstrate the utility and potential of the custom-engineered nanopores by electrical direct single-molecule sensing of 10-nm-sized proteins using widely used research and hand-held analysis devices. The designer nanopores illustrate how DNA nanotechnology can deliver functional biomolecular structures to be used in synthetic biology, single-molecule enzymology and biophysical analysis, as well as portable diagnostics and environmental screening.
Nanion's Orbit 16 TC platform takes the pain out of bilayer painting and subsequent recordings as it enables fast and successful data generation thanks to the rapid automated formation of 16 bilayers at once and their subsequent fully parallel recording.
Bilayer recording is a well-established technique for in-depth studies of biophysical properties of ion channels and is particularly suited for functional studies on proteins residing in intracellular membranes. Moreover, this technique supports a host of powerful emerging analytical techniques using biological nanopores as molecular sensors. Despite its proven value, bilayer recording can be very frustrating due to the capricious nature of lipid bilayers, which have to be formed manually one by one and which often lack stability. We here show a new approach and device, which speeds up the entire process by the rapid and simultaneous formation of 16, highly stable micrometer-sized bilayers using microelectrode cavity array (MECA) chips. A study will be presented showing that the MECA supports high-resolution polymer sizing with a single biological nanopore in a parallel format.
The ongoing pandemic caused by the novel coroNaVirus (SARS-CoV-2) has led to more than 445 million infections and the underlying disease, COVID-19, resulted in more than 6 million deaths worldwide. The scientific world is already predicting future zoonotic diseases. Hence, rapid response systems are needed to tackle future epidemics and pandemics. Here, we present the use of eukaryotic cell-free systems for the rapid response to novel zoonotic diseases represented by SARS-CoV-2. Non-structural, structural and accessory proteins encoded by SARS-CoV-2 were synthesized by cell-free protein synthesis in a fast and efficient manner. The inhibitory effect of the non-structural protein 1 on protein synthesis could be shown in vitro. Structural proteins were quantitatively detected by commercial antibodies, therefore facilitating cell-free systems for the validation of available antibodies. The cytotoxic envelope protein was characterized in electrophysiological planar lipid bilayer measurements. Hence, our study demonstrates the potential of eukaryotic cell-free systems as a rapid response mechanism for the synthesis, functional characterization and antibody validation against a viral pathogen.
Posttranslational modifications (PTMs) of proteins are crucial for cellular function but pose analytical problems, especially in distinguishing chemically identical PTMs at different nearby locations within the same protein. Current methods, such as liquid chromatography-tandem mass spectrometry, are technically tantamount to de novo protein sequencing. Nanopore techniques may provide a more efficient solution, but applying the concepts of nanopore DNA strand sequencing to proteins still faces fundamental problems. Here, we demonstrate the use of an engineered biological nanopore to differentiate positional isomers resulting from acetylation or methylation of histone protein H4, an important PTM target. In contrast to strand sequencing, we differentiate positional isomers by recording ionic current modulations resulting from the stochastic entrapment of entire peptides in the pore’s sensing zone, with all residues simultaneously contributing to the electrical signal. Molecular dynamics simulations show that, in this whole-molecule sensing mode, the non-uniform distribution of the electric potential within the nanopore makes the added resistance contributed by a PTM dependent on its precise location on the peptide. Optimization of the pore’s sensitivity in combination with parallel recording and automated and standardized protein fragmentation may thus provide a simple, label-free, high-throughput analytical platform for identification and quantification of PTMs.
In 2016, the first peptide toxin in any human fungal pathogen was identified. It was discovered in Candida albicans and was named candidalysin. Candidalysin is an amphipathic cationic peptide that damages cell membranes. Like most lytic peptides, candidalysin shows alpha-helical secondary structure. As the helicity and the membrane lytic activity of candidalysin are key factors for pathogenicity, here we describe in vitro approaches to monitor both its membrane-lytic function and the secondary structure. First, membrane permeabilization activity of candidalysin is measured in real time by direct electrical recording. Second, the secondary structure and helicity of candidalysin are determined by circular dichroism spectroscopy. These biophysical methods provide a means to characterize the activity and physical properties of candidalysin in vitro and will be useful in determining the structural and functional features of candidalysin and other similar cationic membrane-active peptides.
The chemical nature and precise position of posttranslational modifications (PTMs) in proteins or peptides are crucial for various severe diseases, such as cancer. State-of-the-art PTM diagnosis is based on elaborate and costly mass-spectrometry or immunoassay-based approaches, which are limited in selectivity and specificity. Here, we demonstrate the use of a protein nanopore to differentiate peptides─derived from human histone H4 protein─of identical mass according to the positions of acetylated and methylated lysine residues. Unlike sequencing by stepwise threading, our method detects PTMs and their positions by sensing the shape of a fully entrapped peptide, thus eliminating the need for controlled translocation. Molecular dynamics simulations show that the sensitivity to molecular shape derives from a highly nonuniform electric field along the pore. This molecular shape-sensing principle offers a path to versatile, label-free, and high-throughput characterizations of protein isoforms.
Single-entity electrochemistry allows the ultrasensitive detection of individual entities by incorporating high bandwidth electrochemical instruments. However, differences among instruments and shielding setups cause large deviations in the recorded signals, which leads to remarkable measurement errors in practical applications. Here, we developed a two-step method to calibrate instruments differences for achieving an accurate single-entity analysis. Two coefficients C1 and C2 were calculated with standard measurement through the model resistors of 1.00 GΩ. C1 obtained from the slope value of the current-voltage (I-V) curve is used to calibrate the shifting of the current statistic distribution. Then, C2, assessed from the standard deviation (STD) noises, is employed to calibrate the full-width half maximum of current statistic distributions. After applying in the model single-molecule experiments of Poly(dA)4 detection with aerolysin nanopores, we showed the effective calibration of measurement differences among four high bandwidth electrochemical instruments. Therefore, this method is generally applicable to all kinds of single-entity electrochemical measurements to reduce measurement errors from instrument differences.
Candidalysin is the first cytolytic peptide toxin identified in any human fungal pathogen. Candidalysin is secreted by Candida albicans and is critical for driving infection and host immune responses in several model systems. However, Candida infections are also caused by non-C. albicans species. Here, we identify and characterize orthologs of C. albicans candidalysin in C. dubliniensis and C. tropicalis. The candidalysins have different amino acid sequences, are amphipathic, and adopt a predominantly α-helical secondary structure in solution. Comparative functional analysis demonstrates that each candidalysin causes epithelial damage and calcium influx and activates intracellular signaling pathways and cytokine secretion. Importantly, C. dubliniensis and C. tropicalis candidalysins have higher damaging and activation potential than C. albicans candidalysin and exhibit more rapid membrane binding and disruption, although both fungal species cause less damage to epithelial cells than C. albicans. This study identifies the first family of peptide cytolysins in human-pathogenic fungi.
Divalent ions are known to have a severe effect on the translocation of several antibiotic molecules into (pathogenic) bacteria. In the present study we have investigated the effect of divalent ions on the permeability of norfloxacin across the major outer membrane channels from E. coli (OmpF and OmpC) and E. aerogenes (Omp35 and Omp36) at the single channel level. To understand the rate limiting steps in permeation, we reconstituted single porins into planar lipid bilayers and analyzed the ion current fluctuations caused in the presence of norfloxacin. Moreover, to obtain an atomistic view, we complemented the experiments with millisecond-long free energy calculations based on temperature-accelerated Brownian dynamics simulations to identify the most probable permeation pathways of the antibiotics through the respective pores. Both, the experimental analysis and the computational modelling, suggest that norfloxacin is able to permeate through the larger porins, i.e., OmpF, OmpC, and Omp35, whereas it only binds to the slightly narrower porin Omp36. Moreover, divalent ions can bind to negatively charged residues inside the porin, reversing the ion selectivity of the pore. In addition, the divalent ions can chelate with the fluoroquinolone molecules and alter their physicochemical properties. The results suggest that the conjugation with either pores or molecules must break when the antibiotic molecules pass the lumen of the porin, with the conjugation to the antibiotic being more stable than that to the respective pore. In general, the permeation or binding process of fluoroquinolones in porins occurs irrespective of the presence of divalent ions, but the presence of divalent ions can vary the kinetics significantly. Thus, a detailed investigation of the interplay of divalent ions with antibiotics and pores is of key importance in developing new antimicrobial drugs.
Efforts to sequence single protein molecules in nanopores have been hampered by the lack of techniques with sufficient sensitivity to discern the subtle molecular differences among all twenty amino acids. Here we report ionic current detection of all twenty proteinogenic amino acids in an aerolysin nanopore with the help of a short polycationic carrier. Application of molecular dynamics simulations revealed that the aerolysin nanopore has a built-in single-molecule trap that fully confines a polycationic carrier-bound amino acid inside the sensing region of the aerolysin. This structural feature means that each amino acid spends sufficient time in the pore for sensitive measurement of the excluded volume of the amino acid. We show that distinct current blockades in wild-type aerolysin can be used to identify 13 of the 20 natural amino acids. Furthermore, we show that chemical modifications, instrumentation advances and nanopore engineering offer a route toward identification of the remaining seven amino acids. These findings may pave the way to nanopore protein sequencing.
Lipid bilayer membranes formed from the artificial 1,3-diamidophospholipid Pad-PC-Pad have the remarkable property that their hydrophobic thickness can be modified in situ: the particular arrangement of the fatty acid chains in Pad-PC-Pad allows them to fully interdigitate below 37 °C, substantially thinning the membrane with respect to the noninterdigitated state. Two stimuli, traversing the main phase transition temperature of the lipid or addition of cholesterol, have previously been shown to disable the interdigitated state. Both manipulations cause an increase in hydrophobic thickness of about 25 Å due to enhanced conformational entropy of the lipids. Here, we characterize the interdigitated state using electrophysiological recordings from free-standing lipid-membranes formed on micro structured electrode CaVity arrays. Compared to standard membranes made from 1,2-diphytanoyl-sn-glycero-3-phosphocholin (DPhPC), pure Pad-PC-Pad membranes at room temperature had lowered electroporation threshold and higher capacitance. Ion channel formation by the peptide Gramicidin A was clearly facilitated in pure Pad-PC-Pad membranes at room temperature, with activity occurring at significantly lower peptide concentrations and channel dwell times increased by 2 orders of magnitude with respect to DPhPC-membranes. Both elevation of temperature beyond the phase transition and addition of cholesterol reduced channel dwell times, as expected if the reduced membrane thickness stabilized channel formation due to decreased hydrophobic mismatch.
Multidrug-resistant bacteria are a great concern and a problem that must be addressed. Extended-spectrum β-lactamases are a common defence mechanism of bacteria to make β-lactam (BL) antibiotics ineffective. β-Lactamase inhibitors (BLIs) are consequently designed and are often clinically prescribed with a BL antibiotic to hinder degradation. Current studies focusing on how BL antibiotics or BLIs interact solely with the bacterial outer membrane nanopores (porins) on reaching the periplasmic side using a nanopore-based sensing technique. In electrochemical studies, the bias voltage allows real-time monitoring of BL antibiotics, BLIs and their mixture through the porin pathway at the single-molecule level. Here we consider the most abundant membrane protein from Escherichia coli (i.e. OmpF), purify and reconstitute the membrane protein in an artificial lipid bilayer and then study its ex vivo electrochemical behaviour. We show the piperacillin/tazobactam mixture interacts with OmpF, whereas the substrate interacts under the maximum bandwidth. The power spectrum analysis of the ionic current trace demonstrates the ampicillin/sulbactram mixture requires more energy than ampicillin alone to pass through the porin pathway. Our results demonstrate that clinically relevant combinations (e.g. piperacillin/tazobactam and ampicillin/sulbactam) interact more strongly with OmpF than either the BL antibiotic or the BLI alone. We suggest a quick and relatively cheap screening method to test the ability of BL antibiotics/BLIs to cross the bacterial cellular membrane.
Cyclic β-sheet decapeptides from the tyrocidine group and the homologous gramicidin S were the first commercially used antibiotics, yet it remains unclear exactly how they kill bacteria. We investigated their mode of action using a bacterial cytological profiling approach. Tyrocidines form defined ion-conducting pores, induce lipid phase separation, and strongly reduce membrane fluidity, resulting in delocalization of a broad range of peripheral and integral membrane proteins. Interestingly, they also cause DNA damage and interfere with DNA-binding proteins. Despite sharing 50% sequence identity with tyrocidines, gramicidin S causes only mild lipid demixing with minor effects on membrane fluidity and permeability. Gramicidin S delocalizes peripheral membrane proteins involved in cell division and cell envelope synthesis but does not affect integral membrane proteins or DNA. Our results shed a new light on the multifaceted antibacterial mechanisms of these antibiotics and explain why resistance to them is virtually nonexistent. Importance:Cyclic β-sheet decapeptides, such as tyrocidines and gramicidin S, were among the first antibiotics in clinical application. Although they have been used for such a long time, there is virtually no resistance to them, which has led to a renewed interest in this peptide class. Both tyrocidines and gramicidin S are thought to disrupt the bacterial membrane. However, this knowledge is mainly derived from in vitro studies, and there is surprisingly little knowledge about how these long-established antibiotics kill bacteria. Our results shed new light on the antibacterial mechanism of β-sheet peptide antibiotics and explain why they are still so effective and why there is so little resistance to them.
Proteinaceous nanometer-scale pores have been used to detect and physically characterize many different types of analytes at the single-molecule limit. The method is based on the ability to measure the transient reduction in the ionic channel conductance caused by molecules that partition into the pore. The distribution of blockade depth amplitudes and residence times of the analytes in the pore are used to physically and chemically characterize them. Here we compare the current blockade events caused by flexible linear polymers of ethylene glycol (PEGs) and structurally well-defined tungsten polyoxymetallate nanoparticles in the nanopores formed by Staphylococcus aureus α -hemolysin and Aeromonas hydrophila aerolysin. Surprisingly, the variance in the ionic current blockade depth values for the relatively rigid metallic nanoparticles is much greater than that for the flexible PEGs, possibly because of multiple charged states of the polyoxymetallate clusters.
Small, hydrophilic molecules, including most important antibiotics in clinical use, cross the Gram-negative outer membrane through the water-filled channels provided by porins. We have determined the X-ray crystal structures of the principal general porins from three species of Enterobacteriaceae, namely Enterobacter aerogenes, Enterobacter cloacae and Klebsiella pneumoniae and determined their antibiotic permeabilities as well as those of the orthologues from Escherichia coli. Starting from the structure of the porins and molecules we propose a physical mechanism underlying transport and condense it in a computationally efficient scoring function. The scoring function shows good agreement with in-vitro penetration data and will enable the screening of virtual databases to identify molecules with optimal permeability through porins and help to guide the optimization of antibiotics with poor permeation.
We use two pore-forming proteins, alpha-hemolysin and aerolysin, to compare the polymer size-dependence of ionic current block by two types of ethyleneglycol polymers: 1) linear and 2) 3-arm star poly(ethylene glycol), both applied as a polydisperse mixture of average mass 1kDa under high salt conditions. The results demonstrate that monomer size sensitivity, as known for linear PEGs, is conserved for the star polymers with only subtle differences in the dependence of the residual conductance on monomer number. To explain this absence of a dominant effect of polymer architecture, we propose that PEG adsorbs to the inner pore wall in a collapsed, salted-out state, likely due to the effect of hydrophobic residues in the pore wall on the availability of water for hydration.
The pore forming characteristic of TDH1 and TDH2 variants of thermostable direct hemolysin (TDH), a major toxin involved in the pathogenesis of Vibrio parahaemolyticus, was studied on a planar lipid bilayer painted over individual picoliter CaVities containing microelectrodes assembled in a multiarray. Both proteins formed pores upon insertion into the lipid bilayer which was shown as a shift in the conductance from the baseline current. TDH2 protein was able to produce stable currents and the currents were influenced by external factors like concentration, type of salt and voltage. The pore currents were influenced and showed a detectable response in the presence of polymers which makes them suitable for biotechnology applications.
DNA nanopores are a recent class of bilayer-puncturing nanodevices that can help advance biosensing, synthetic biology, and nanofluidics. Here, we create archetypal lipid-anchored DNA nanopores and characterize them with a nanoprobe-based approach to gain essential information about their interactions with bilayers. The strategy determines the molecular accessibility of DNA pores with a nuclease and can thus distinguish between the nanopores' membrane-adhering and membrane-spanning states. The analysis reveals, for example, that pores interact with bilayers in two steps whereby fast initial membrane tethering is followed by slower reorientation to the puncturing state. Tethering occurs for pores with one anchor, while puncturing requires multiple anchors. Both low and high-curvature membranes are good substrates for tethering, but efficient insertion proceeds only for high-curvature bilayers of the examined lipid composition. This is likely due to the localized lipid misalignments and the associated lower energetic barrier for pore permeation. Our study advances the fields of DNA nanotechnology and nanopores by overcoming the considerable experimental hurdle of efficient membrane insertion. It also provides mechanistic insights to aid the design of advanced nanopores, and offers a useful route to probe bilayer orientation of DNA nanostructures.
Recently developed DNA-based analogues of membrane proteins have advanced synthetic biology. A fundamental question is how hydrophilic nanostructures reside in the hydrophobic environment of the membrane. Here, we use multiscale molecular dynamics (MD) simulations to explore the structure, stability and dynamics of an archetypical DNA nanotube inserted via a ring of membrane anchors into a phospholipid bilayer. Coarse-grained MD reveals that the lipids reorganize locally to interact closely with the membrane-spanning section of the DNA tube. Steered simulations along the bilayer normal establish the metastable nature of the inserted pore, yielding a force profile with barriers for membrane exit due to the membrane anchors. Atomistic, equilibrium simulations at two salt concentrations confirm the close packing of lipid around of the stably inserted DNA pore and its cation selectivity, while revealing localized structural fluctuations. The wide-ranging and detailed insight informs the design of next-generation DNA pores for synthetic biology or biomedicine.
Bacillus thuringiensis parasporal crystal proteins (Cry proteins) are insecticidal pore-forming toxins that bind to specific receptor molecules on the brush border membrane of susceptible insect midgut cells to exert their toxic action. In the Colorado potato beetle (CPB), a coleopteran pest, we previously proposed that interaction of Cry3Aa toxin with a CPB ADAM10 metalloprotease is an essential part of the mode of action of this toxin. Here, we annotated the gene sequence encoding an ADAM10 metalloprotease protein (CPB-ADAM10) in the CPB genome sequencing project, and using RNA interference gene silencing we demonstrated that CPB-ADAM10 is a Cry3Aa toxin functional receptor in CPB. Cry3Aa toxicity was significantly lower in CPB-ADAM10 silenced larvae and in vitro toxin pore-forming ability was greatly diminished in lipid planar bilayers fused with CPB brush border membrane vesicles (BBMVs) prepared from CPB-ADAM10 silenced larvae. In accordance with our previous data that indicated this toxin was a substrate of ADAM10 in CPB, Cry3Aa toxin membrane-associated proteolysis was altered when CPB BBMVs lacked ADAM10. The functional validation of CPB-ADAM10 as a Cry3Aa toxin receptor in CPB expands the already recognized role of ADAM10 as a pathogenicity determinant of pore-forming toxins in humans to an invertebrate species.
The 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.
Cell-free protein synthesis (CFPS) represents a promising technology for efficient protein production targeting especially so called “difficult-to-express” proteins whose synthesis is challenging in conventional in vivo protein production platforms. Chinese hamster ovary (CHO) cells are one of the most prominent and safety approved cell lines for industrial protein production. In this study we demonstrated the ability to produce high yields of various protein types including membrane proteins and single chain variable fragments (scFv) in a continuous exchange cell-free (CECF) system based on CHO cell lysate that contains endogenous microsomal structures. We showed significant improvement of protein yield compared to batch formatted reactions and proved biological activity of synthesized proteins using various analysis technologies. Optimized CECF reaction conditions led to membrane protein yields up to 980 µg/ml, which is the highest protein yield reached in a microsome containing eukaryotic cell-free system presented so far.
Biological ion channels are molecular gatekeepers that control transport across cell membranes. Recreating the functional principle of such systems and extending it beyond physiological ionic cargo is both scientifically exciting and technologically relevant to sensing or drug release. However, fabricating synthetic channels with a predictable structure remains a significant challenge. Here, we use DNA as a building material to create an atomistically determined molecular valve that can control when and which cargo is transported across a bilayer. The valve, which is made from seven concatenated DNA strands, can bind a specific ligand and, in response, undergo a nanomechanical change to open up the membrane-spanning channel. It is also able to distinguish with high selectivity the transport of small organic molecules that differ by the presence of a positively or negatively charged group. The DNA device could be used for controlled drug release and the building of synthetic cell-like or logic ionic networks
Alamethicins (ALMs) are antimicrobial peptides of fungal origin. Their sequences are rich in hydrophobic amino acids and strongly interact with lipid membranes, where they cause a well-defined increase in conductivity. Therefore, the peptides are thought to form transmembrane helical bundles in which the more hydrophilic residues line a water-filled pore. Whereas the peptide has been well characterized in terms of secondary structure, membrane topology, and interactions, much fewer data are available regarding the quaternary arrangement of the helices within lipid bilayers. A new, to our knowledge, fluorine-labeled ALM derivative was prepared and characterized when reconstituted into phospholipid bilayers. As a part of these studies, C19F3-labeled compounds were characterized and calibrated for the first time, to our knowledge, for 19F solid-state NMR distance and oligomerization measurements by centerband-only detection of exchange (CODEX) experiments, which opens up a large range of potential labeling schemes. The 19F-19F CODEX solid-state NMR experiments performed with ALM in POPC lipid bilayers and at peptide/lipid ratios of 1:13 are in excellent agreement with molecular-dynamics calculations of dynamic pentameric assemblies. When the peptide/lipid ratio was lowered to 1:30, ALM was found in the dimeric form, indicating that the supramolecular organization is tuned by equilibria that can be shifted by changes in environmental conditions.
Curli are functional amyloid fibres that constitute the major protein component of the extracellular matrix in pellicle biofilms formed by Bacteroidetes and Proteobacteria (predominantly of the α and γ classes). They provide a fitness advantage in pathogenic strains and induce a strong pro-inflammatory response during bacteraemia. Curli formation requires a dedicated protein secretion machinery comprising the outer membrane lipoprotein CsgG and two soluble accessory proteins, CsgE and CsgF. Here we report the X-ray structure of Escherichia coli CsgG in a non-lipidated, soluble form as well as in its native membrane-extracted conformation. CsgG forms an oligomeric transport complex composed of nine anticodon-binding-domain-like units that give rise to a 36-stranded β-barrel that traverses the bilayer and is connected to a cage-like vestibule in the periplasm. The transmembrane and periplasmic domains are separated by a 0.9-nm channel constriction composed of three stacked concentric phenylalanine, asparagine and tyrosine rings that may guide the extended polypeptide substrate through the secretion pore. The specificity factor CsgE forms a nonameric adaptor that binds and closes off the periplasmic face of the secretion channel, creating a 24,000 Å3 pre-constriction chamber. Our structural, functional and electrophysiological analyses imply that CsgG is an ungated, non-selective protein secretion channel that is expected to employ a diffusion-based, entropy-driven transport mechanism.
Efficient use of membrane protein nanopores in ionic single-molecule sensing requires technology for the reliable formation of suspended molecular membranes densely arrayed in formats that allow high-resolution electrical recording. Here, automated formation of bimolecular lipid layers is shown using a simple process where a poly(tetrafluoroethylene)-coated magnetic bar is remotely actuated to perform a turning motion, thereby spreading phospholipid in organic solvent on a nonpolar surface containing a 1 mm2 4 × 4 array of apertures with embedded microelectrodes (microelectrode CaVity array). Parallel and high-resolution single-molecule detection by single nanopores is demonstrated on the resulting bilayer arrays, which are shown to form by a classical but very rapid self-assembly process. The technique provides a robust and scalable solution for the problem of reliable, automated formation of multiple independent lipid bilayers in a dense microarray format, while preserving the favorable electrical properties of the microelectrode CaVity array.
DNA nanotechnology excels at rationally designing bottom-up structures that can functionally replicate naturally occurring proteins. Here we describe the design and generation of a stable DNA-based nanopore that structurally mimics the amphiphilic nature of protein pores and inserts into bilayers to support a steady transmembrane flow of ions. The pore carries an outer hydrophobic belt comprised of small chemical alkyl groups which mask the negatively charged oligonucleotide backbone. This modification overcomes the otherwise inherent energetic mismatch to the hydrophobic environment of the membrane. By merging the fields of nanopores and DNA nanotechnology, we expect that the small membrane-spanning DNA pore will help open up the design of entirely new molecular devices for a broad range of applications including sensing, electric circuits, catalysis, and research into nanofluidics and controlled transmembrane transport.
High throughput and a long life-time of the devices are two crucial challenges in planar chip technology for electrophysiological measurements of ionic current recording through ion channel proteins. In this paper, we present a wafer-scale process for the generation of novel arrays of microelectrochemical cells for long-term and high-resolution current recording. At the bottom of each of the cells, which have typically diameters of around 60 μm and volumes of around 30 pL, a nanocrystalline silver/silver chloride secondary electrode is generated for ionic current recording. The top of the cell is closed by a lid containing a small (6–16 μm) opening which connects liquid in the chamber to a contacting liquid on the outside. The processes necessary for manufacturing such a chip through photolithography and wafer-scale bonding have been developed, the resulting structures were characterized and the procedures were optimized. Combining a large surface area of the electrode with a – in relation to the cell size – relatively large amount of silver/silver chloride allows for the recording of DC ionic currents for prolonged periods of time. First measurements were performed where the electrochemical cells were closed by model membranes containing single ion channel proteins. The currents generated by ions passing through these ion channels are reported. These measurements demonstrate the usefulness of the microelectrochemical cell array for long time ionic current recordings at – for these type of measurements – relatively high current levels.
We created nanometer-scale transmembrane channels in lipid bilayers using self-assembled DNA-based nanostructures. Scaffolded DNA origami was used to create a stem that penetrates and spans a lipid membrane, and a barrel-shaped cap that adheres to the membrane in part via 26 cholesterol moieties. In single-channel electrophysiological measurements, we find similarities to the response of natural ion channels, such as conductances on the order of 1 nS and channel gating. More pronounced gating was seen for mutations in which a single DNA strand of the stem protruded into the channel. In single-molecule translocation experiments, we highlight one of many potential applications of the synthetic channels, namely as single DNA molecule sensing devices.
We report on parallel high-resolution electrical single-molecule analysis on a chip-based nanopore microarray. Lipid bilayers of 20 μm diameter containing single alpha-hemolysin pores were formed on arrays of subpicoliter CaVities containing individual microelectrodes (microelectrode CaVity array, MECA), and ion conductance-based single molecule mass spectrometry was performed on mixtures of poly(ethylene glycol) molecules of different length. We thereby demonstrate the function of the MECA device as a chip-based platform for array-format nanopore recordings with a resolution at least equal to that of established single microbilayer supports. We conclude that devices based on MECAs may enable more widespread analytical use of nanopores by providing the high throughput and ease of operation of a high-density array format while maintaining or exceeding the precision of state-of-the-art microbilayer recordings.
Increasing the throughput and resolution of electrical recording of currents through ion conducting channels and pores is an important technical challenge both for the functional analysis of ion channel proteins and for the application of nanoscale pores in single molecule analytical tasks. We present a novel design based on sub-picoliter-CaVities arrayed in a polymer substrate and endowed with individual planar microelectrodes that allows low-noise and parallel electrical recording from ion channels and pores. Resolution of voltage-dependent current transitions of alamethicin channels as well as polyethylene-glycol-induced blocking events of alpha-hemolysin nanopores on the submillisecond time scale is demonstrated using this device.
