Protein Nanopore Research Articles

Insertion state of modular protein nanopores into a membrane

In the past decade, significant progress has been made in the development of new protein nanopores. Despite these advancements, there is a pressing need for the creation of nanopores equipped with relatively large functional groups for the sampling of biomolecular events on their extramembranous side. Here, we designed, produced, and analyzed protein nanopores encompassing a robust truncation of a monomeric β-barrel membrane protein. An exogenous stably folded protein was anchored within the aqueous phase via a flexible peptide tether of varying length. We have extensively examined the pore-forming properties of these modular protein nanopores using protein engineering and single-molecule electrophysiology. This study revealed distinctions in the nanopore conductance and current fluctuations that arose from tethering the exogenous protein to either the N terminus or the C terminus. Remarkably, these nanopores insert into a planar lipid membrane with one specific conductance among a set of three substate conductance values. Moreover, we demonstrate that the occurrence probabilities of these insertion substates depend on the length of the peptide tether, the orientation of the exogenous protein with respect to the nanopore opening, and the molecular mass of tethered protein. In addition, the three conductance values remain unaltered by major changes in the composition of modular nanopores. The outcomes of this work serve as a platform for further developments in areas of protein engineering of transmembrane pores and biosensor technology.

Tertiary RNA Folding-Targeted Drug Screening Strategy Using a Protein Nanopore.

Bacterial riboswitch RNAs are attractive targets for novel antibiotics against antibiotic-resistant superbacteria. Their binding to cognate metabolites is essential for the regulation of bacterial gene expression. Despite the importance of RNAs as therapeutic targets, the development of RNA-targeted, small-molecule drugs is limited by current biophysical methods. Here, we monitored the specific interaction between the adenine-sensing riboswitch aptamer domain (ARS) and adenine at the single-molecule level using α-hemolysin (αHL) nanopores. During adenine-induced tertiary folding, adenine-bound ARS intermediates exhibited characteristic nanopore events, including a two-level ionic current blockade and a ∼ 5.6-fold longer dwell time than that of free RNA. In a proof-of-concept experiment, tertiary RNA folding-targeted drug screening was performed using a protein nanopore, which resulted in the discovery of three new ARS-targeting hit compounds from a natural compound library. Taken together, these results reveal that αHL nanopores are a valuable platform for ultrasensitive, label-free, and single-molecule-based drug screening against therapeutic RNA targets.

Simultaneous Sensing of Force and Current Signals to Recognize Proteinogenic Amino Acids at a Single-Molecule Level.

The identification ability of nanopore sequencing is severely hindered by the diversity of amino acids in a protein. To tackle this problem, a graphene nanoslit sensor is adopted to collect force and current signals to distinguish 20 residues. Extensive molecular dynamics simulations are performed on sequencing peptides under pulling force and applied electric field. Results show that the signals of force and current can be simultaneously collected. Tailoring the geometry of the nanoslit sensor optimizes signal differences between tyrosine and alanine residues. Using the tailored geometry, the characteristic signals of 20 types of residues are detected, enabling excellent distinguishability so that the residues are well-grouped by their properties and signals. The signals reveal a trend in which the larger amino acids have larger pulling forces and lower ionic currents. Generally, the graphene nanoslit sensor can be employed to simultaneously sense two signals, thereby enhancing the identification ability and providing an effective mode of nanopore protein sequencing.

Mapping the morphological identifiers of distinct conformations via the protein translocation current in nanopores

Conformational changes of proteins play a vital role in implementing their functions and revealing the underlying mechanisms in various biological processes. It is still challenging to monitor protein conformations with temporal fingerprints of current-resistance pulses in the nanopore technique. Here the low-resolution morphologies of different conformations of a typical integrin, αxβ2, were estimated via relative blockade currents simulated from all-atom molecular dynamics (MD). Distinct conformational states of αxβ2 were directly explained by the volume and shape identifiers. Protein modulation in ionic current was analyzed from the conductivity distribution inside the protein-blocked nanopore. Combining a discrete model with spheroidal approximation, a MD-based approach was developed to theoretically predict the volume and shape of the nanopore for sensing αxβ2. This method was also applicable in specifying morphological identifiers of six other proteins, and the theoretical predictions are in good agreement with the experimental measurements. These results potentiated the validity of this method for the conformational identification of proteins in nanopores.

Measuring interactions of DNA with nanoporous protein crystals by atomic force microscopy.

Crosslinked porous protein crystals are a new biomaterial that can be engineered to encapsulate, stabilize, and organize guest molecules, nanoparticles, and biological moieties. In this study, for the first time, the combined interactions of DNA strands with porous protein crystals are quantitatively measured by high-resolution atomic force microscopy (AFM) and chemical force microscopy. The surface structure of protein crystals with unusually large pores was observed in liquid via high-resolution AFM. Force-distance (F-D) curves were also obtained using AFM tips modified to present or capture DNA. The modification of AFM tips allowed the tips to covalently bind DNA that was pre-loaded in the protein crystal nanopores. The modified tips enabled the interactions of DNA molecules with protein crystals to be quantitatively studied while revealing the morphology of the buffer-immersed protein crystal surface in detail, thereby preserving the structure and properties of protein crystals that could be disrupted or destroyed by drying. The hexagonal space group was manifest at the crystal surface, as were the strong interactions between DNA and the porous protein crystals in question. In sum, this study furthered our understanding of how a new protein-based biomaterial can be used to bind guest DNA assemblies.

A Lipid-Bilayer-On-A-Cup Device for Pumpless Sample Exchange.

Lipid-bilayer devices have been studied for on-site sensors in the fields of diagnosis, food and environmental monitoring, and safety/security inspection. In this paper, we propose a lipid-bilayer-on-a-cup device for serial sample measurements using a pumpless solution exchange procedure. The device consists of a millimeter-scale cylindrical cup with vertical slits which is designed to steadily hold an aqueous solution and exchange the sample by simply fusing and splitting the solution with an external solution. The slit design was experimentally determined by the capabilities of both the retention and exchange of the solution. Using the optimized slit, a planar lipid bilayer was reconstituted with a nanopore protein at a microaperture allocated to the bottom of the cup, and the device was connected to a portable amplifier. The solution exchangeability was demonstrated by observing the dilution process of a blocker molecule of the nanopore dissolved in the cup. The pumpless solution exchange by the proposed cup-like device presents potential as a lipid-bilayer system for portable sensing applications.

Protein Ligand-Induced Amplification in the 1/f Noise of a Protein-Selective Nanopore.

Previous studies of transmembrane protein channels have employed noise analysis to examine their statistical current fluctuations. In general, these explorations determined a substrate-induced amplification in the Gaussian white noise of these systems at a low-frequency regime. This outcome implies a lack of slowly appearing fluctuations in the number and local mobility of diffusing charges in the presence of channel substrates. Such parameters are among the key factors in generating a low-frequency 1/f noise. Here, we show that a protein-selective biological nanopore exhibits a substrate-induced amplification in the 1/f noise. The modular composition of this biological nanopore includes a hydrophilic transmembrane protein pore fused to a water-soluble binding protein on its extramembranous side. In addition, this protein nanopore shows an open substate populated by a high-frequency current noise because of the flickering of an engineered polypeptide adaptor at the tip of the pore. However, the physical association of the protein ligand with the binding domain reversibly switches the protein nanopore from a high-frequency noise substate into a quiet substate. In the absence of the protein ligand, our nanopore shows a low-frequency white noise. Remarkably, in the presence of the protein ligand, an amplified low-frequency 1/f noise was detected in a ligand concentration-dependent fashion. This finding suggests slowly occurring equilibrium fluctuations in the density and local mobility of charge carriers under these conditions. Furthermore, we report that the excess in 1/f noise is generated by reversible switches between the noisy ligand-released substate and the quiet ligand-captured substate. Finally, quantitative aspects of the low-frequency 1/f noise are in accord with theoretical predictions of the current noise analysis of protein channel-ligand interactions.

Non-Receptor-Mediated Lipid Membrane Permeabilization by the SARS-CoV-2 Spike Protein S1 Subunit.

Due to the pressing need to generate specific drugs or vaccines for COVID-19 and management of its outbreak, detailed knowledge regarding the SARS-CoV-2 entry into host cells and timely, cheap, and easy-to-use detection methods are of critical importance for containing the SARS-CoV-2 epidemic. Through electrophysiology and fluorescence spectroscopy experiments, we show that even in the absence of the angiotensin-converting enzyme 2 receptor, the S1 subunit from SARS-CoV-2 spike protein binding to neutral phospholipid membranes leads to their mechanical destabilization and permeabilization. A similar cytotoxic effect of the protein was seen in human lung epithelial cells. A monoclonal antibody generated toward the S1 subunit alleviates to a considerable extent the destabilizing potential of the protein in such model membranes. Finally, we demonstrate the proof-of-concept capability of an α-hemolysin (α-HL) protein nanopore to detect in aqueous buffer and real time the region-binding domain of the S1 subunit from SARS-CoV-2 spike protein by monitoring its immunological interaction with a target antibody. Our results may offer new perspectives in understanding the pathogenesis of the SARS-CoV-2 infection, its treatment, and real-time detection.

A review on the role of emerging revolutionary nanotechnology in forensic investigations

Due to the unique properties of nanoparticles, it has gained prominence in lots of fields with extensive research being carried around it. With lots of novel applications arising from this field, Forensic science seems to be one of the fast-growing fields in nano research applications. The growing and extensive use of nanotechnology being applied in forensic investigations is promising and could soon be the tipping point in the discipline. Applications mainly have been related to evidence identification and analysis in the broad major fields in Forensic Science such as single-crystalline semiconductor CdS nano slabs for explosives detection, functionalized TiO2 nanorods for organophosphorus chemical warfare agents in Forensic Chemistry, the use of Nanopowders for latent print visualization in Forensic physics and Gold nanoparticle protein nanopore for detection of single-stranded DNA in Forensic biology. Nanotechnology has also been employed in illegal drug detection in recent times. These and other applications of Nanotechnology provides prompt and precise results with reduced methods due to the limited instruments used for analyzing evidence as well as providing sensitive and selective ways of detecting evidence. As evidence is notable in forensic investigations, nanotechnology’s use in identifying and detecting these has potential in enhancing and providing efficient and rapid means for investigations and unravelling leads into crimes. This review emphasizes some disciplines in forensic sciences in which nanotechnology is having an impact, novel methods and newly developed instruments and also takes into account its challenges as well as perspectives into the future.

Exploration of Samples’ Surface Properties: A Review

Scanning ion conductance microscopy (SICM) have attracted great attention as one of the most promising candidates for imaging soft and delicate biological samples. In this review, firstly, up to date research on investigating samples’ surface properties using SICM are demonstrated. In surface charge part, as the bias is varied, a polarity-dependent ion current and surface-induced rectification can help SICM to create images of surface charge. In dynamics of living cells part, SICM allows both non-labeling and non-contact during the measurement and performs the best smong fluorescent microscopy and AFM in visualizing dynamics of living cells. In surface morphology part, four examples are demonstrated to show SICM’s power. Secondly, combinations with mature technology such as scanning electrochemical microscopy (SECM), sub-aperture stitching technology, stimulated emission depletion (STED) microscopy and protein nanopores are present, which achieve the multifunction of SICM. Thirdly, state-of-the-art ways that can improve imaging rate are demonstrated. Finally, a brief future outlook for SICM is discussed.

Probing the Neuraminidase Activity of Influenza Virus Using a Cytolysin A Protein Nanopore.

Neuraminidase (NA), one of the major surface glycoproteins of influenza A virus (IAV), is an important diagnostic biomarker and antiviral therapeutic target. Cytolysin A (ClyA) is a nanopore sensor with an internal constriction of 3.3 nm, enabling the detection of protein conformations at the single-molecule level. In this study, a nanopore-based approach is developed for analysis of the enzymatic activity of NA, which facilitates rapid and highly sensitive diagnosis of IAV. Current blockade analysis of the d-glucose/d-galactose-binding protein (GBP) trapped within a type I ClyA-AS (ClyA mutant) nanopore reveals that galactose cleaved from sialyl-galactose by NA of the influenza virus can be detected in real time and at the single-molecule level. Our results show that this nanopore sensor can quantitatively measure the activity of NA with 40-80-fold higher sensitivity than those previously reported. Furthermore, the inhibition of NA is monitored using small-molecule antiviral drugs, such as zanamivir. Taken together, our results reveal that the ClyA protein nanopore can be a valuable platform for the rapid and sensitive point-of-care diagnosis of influenza and for drug screening against the NA target.

Nanopore Enzymology to Study Protein Kinases and Their Inhibition by Small Molecules.

Nanopore enzymology is a powerful single-molecule technique for the label-free study of enzymes using engineered protein nanopore sensors. The technique has been applied to protein kinases, where it has enabled the full repertoire of kinase function to be observed, including: kinetics of substrate binding and dissociation, product binding and dissociation, nucleotide binding, and reversible phosphorylation. Further, minor modifications enable the screening of type I kinase inhibitors and the determination of inhibition constants in a facile and label-free manner. Here, we describe the design and production of suitably engineered protein nanopores and their use for the determination of key mechanistic parameters of kinases. We also provide procedures for the determination of inhibition constants of protein kinase inhibitors.

Water in Nanopores and Biological Channels: A Molecular Simulation Perspective.

This Review explores the dynamic behavior of water within nanopores and biological channels in lipid bilayer membranes. We focus on molecular simulation studies, alongside selected structural and other experimental investigations. Structures of biological nanopores and channels are reviewed, emphasizing those high-resolution crystal structures, which reveal water molecules within the transmembrane pores, which can be used to aid the interpretation of simulation studies. Different levels of molecular simulations of water within nanopores are described, with a focus on molecular dynamics (MD). In particular, models of water for MD simulations are discussed in detail to provide an evaluation of their use in simulations of water in nanopores. Simulation studies of the behavior of water in idealized models of nanopores have revealed aspects of the organization and dynamics of nanoconfined water, including wetting/dewetting in narrow hydrophobic nanopores. A survey of simulation studies in a range of nonbiological nanopores is presented, including carbon nanotubes, synthetic nanopores, model peptide nanopores, track-etched nanopores in polymer membranes, and hydroxylated and functionalized nanoporous silica. These reveal a complex relationship between pore size/geometry, the nature of the pore lining, and rates of water transport. Wider nanopores with hydrophobic linings favor water flow whereas narrower hydrophobic pores may show dewetting. Simulation studies over the past decade of the behavior of water in a range of biological nanopores are described, including porins and β-barrel protein nanopores, aquaporins and related polar solute pores, and a number of different classes of ion channels. Water is shown to play a key role in proton transport in biological channels and in hydrophobic gating of ion channels. An overall picture emerges, whereby the behavior of water in a nanopore may be predicted as a function of its hydrophobicity and radius. This informs our understanding of the functions of diverse channel structures and will aid the design of novel nanopores. Thus, our current level of understanding allows for the design of a nanopore which promotes wetting over dewetting or vice versa. However, to design a novel nanopore, which enables fast, selective, and gated flow of water de novo would remain challenging, suggesting a need for further detailed simulations alongside experimental evaluation of more complex nanopore systems.

Single-sulfur atom discrimination of polysulfides with a protein nanopore for improved batteries

Research on batteries mostly focuses on electrodes and electrolytes while few activities regard separator membranes. However, they could be used as a toolbox for injecting chemical functionalities to capture unwanted species and enhance battery lifetime. Here, we report the use of biological membranes hosting a nanopore sensor for electrical single molecule detection and use aqueous sodium polysulfides encountered in sulfur-based batteries for proof of concept. By investigating the host-guest interaction between polysulfides of different chain-lengths and cyclodextrins, via combined chemical approaches and molecular docking simulations, and using a selective nanopore sensor inserted into a lipid membrane, we demonstrate that supramolecular polysulfide/cyclodextrin complexes only differing by one sulfur can be discriminated at the single molecule level. Our findings offer innovative perspectives to use nanopores as electrolyte sensors and chemically design membranes capable of selective speciation of parasitic molecules for battery applications and therefore pave the way towards smarter electrochemical storage systems.

A dual-constriction biological nanopore resolves homonucleotide sequences with high fidelity.

Single-molecule long-read DNA sequencing with biological nanopores is fast and high-throughput but suffers reduced accuracy in homonucleotide stretches. We now combine the CsgG nanopore with the 35-residue N-terminal region of its extracellular interaction partner CsgF to produce a dual-constriction pore with improved signal and basecalling accuracy for homopolymer regions. The electron cryo-microscopy structure of CsgG in complex with full-length CsgF shows that the 33 N-terminal residues of CsgF bind inside the β-barrel of the pore, forming a defined second constriction. In complexes of CsgG bound to a 35-residue CsgF constriction peptide, the second constriction is separated from the primary constriction by ~25 Å. We find that both constrictions contribute to electrical signal modulation upon ssDNA translocation. DNA sequencing using a prototype CsgG:CsgF protein pore with two constrictions improved single-read accuracy by 25 to 70 % in homopolymers up to 9 nucleotides long.

Insight into the effects of electrochemical factors on host-guest interaction induced signature events in a biological nanopore.

The signature events caused by host-guest interactions in the nanopore system can be used as a novel and characteristic signal in quantitative detection and analysis of various molecules. However, the effect of several electrochemical factors on the host-guest interactions in nanopore still remains unknown. Here, we systematically studied host-guest interactions, especially oscillation of DNA-azide adamantane@cucurbit[6] in α-Hemolysin nanopore under varying pH, concentration of electrolytes and counterions (Li+, Na+, K+). Our results indicate correlations between the change of pH and the duration of the oscillation signal. In addition, the asymmetric electrolyte concentration and the charge of the counterions affects the frequency of signature events in oscillation signals, and even the integrity of the protein nanopore. This study provides insight into the design of a future biosensing platform based on signature oscillation signals of the host-guest interaction within a nanopore.

Molecular Insights into Distinct Detection Properties of α-Hemolysin, MspA, CsgG, and Aerolysin Nanopore Sensors.

Protein nanopores have been widely used as single-molecule sensors for the detection and characterization of biological polymers such as DNA, RNA, and polypeptides. A variety of protein nanopores with various geometries have been exploited for this purpose, which usually exhibit distinct sensing capabilities, but the underlying molecular mechanism remains elusive. Here, we systematically characterize the molecular transport properties of four widely studied protein nanopores, α-hemolysin, MspA, CsgG, and aerolysin, by extensive molecular dynamics simulations. It is found that a sudden drop in electrostatic potentials occurs at the sole constriction in MspA and CsgG nanopores in contrast to the gradual potential change inside α-hemolysin and aerolysin pores, indicating the crucial role of pore geometry in ionic and molecular transport. We further demonstrate that these protein nanopores exhibit open-pore currents and ssDNA-induced current blockades both in the order MspA > α-hemolysin > CsgG > aerolysin, but an equivalent blockade percentage around 80%. In addition, the substitution of key amino acids at the pore constriction, especially by charged ones, provides an efficient way to modulate the pore electrostatic potential and ionic current. This work sheds new light on the search for high-performance nanopores, engineering of protein nanopores, and design of bioinspired solid-state nanopores.

Crowding-Induced DNA Translocation through a Protein Nanopore.

A crowded cellular environment is highly associated with many significant biological processes. However, the effect of molecular crowding on the translocation behavior of DNA through a pore has not been explored. Here, we use nanopore single-molecule analytical technique to quantify the thermodynamics and kinetics of DNA transport under heterogeneous cosolute PEGs. The results demonstrate that the frequency of the translocation event exhibits a nonmonotonic dependence on the crowding agent size, while both the event frequency and translocation time increase monotonically with increasing crowder concentration. In the presence of PEGs, the rate of DNA capture into the nanopore elevates 118.27-fold, and at the same time the translocation velocity decreases from 20 to 120 μs/base. Interestingly, the impact of PEG 4k on the DNA-nanopore interaction is the most notable, with up to ΔΔG = 16.27 kJ mol-1 change in free energy and 764.50-fold increase in the binding constant at concentration of 40% (w/v). The molecular crowding effect will has broad applications in nanopore biosensing and nanopore DNA sequencing in which the strategy to capture analyte and to control the transport is urgently required.

Nanopore-Based Analysis of Conformational Heterogeneity of Nucleic Acids using a Gamma-Hemolysin Protein Channel

Protein nanopores have been used to stochastically detect and characterize analytes for chemical reactions, biomolecule analysis, or biomedical applications. The detection sensitivity and specificity of the targeted analyte are significantly affected by both physical and chemical interactions between the analyte and the selected protein pore. We have demonstrated that a γ-hemolysin (γ-HL) protein channel (HlgB-HlgC) can assemble into an octameric pore on a planar lipid bilayer, and its dimensionality is wide enough to translocate canonical B-form double-stranded DNA (dsDNA). To broaden the analytical application of this γ-HL protein channel, we explore its ability to sample and distinguish nucleic acid conformational polymorphisms, such as A-, B-, or Z-form helixes, with single-molecule resolution. The canonical B-form structure and the slimmest left-handed Z-form DNA duplexes translocate through the γ-HL nanopore; in contrast, the A-form DNA-RNA hybrid duplex with a wider diameter is unable to translocate through the protein channel until unzipping into its two single-stranded components. The current signatures reveal multiple interactions between the nucleic acids and the γ-HL nanopore. Our results introduce the γ-HL protein channel (HlgB-HlgC) as an analytical tool for nucleic acid analysis at the single-molecule level. Our results demonstrate that this protein nanopore can be used to investigate the conformational heterogeneity and structural transitions of duplex nucleic acids.

Interaction of Cucurbituril Molecular Containers with the Aerolysin Nanopore for Molecular Recognition

Cucurbit[n]urils (CBn) are molecular containers which form complexes with various guest molecules, such as amino acids and peptides. Here we report the ionic current block and reversible binding of different CBn of sizes n=5, n=6, n=7 and n=8 with the aerolysin (Ael) protein nanopore. CBn molecular containers enter the Ael nanopore exclusively from the extracellular cap domain and under a trans negative voltage. We attempt to relate differential effects of the electrolytes KCl, NaCl, CsCl and LiCl and of the ionic strength on the voltage-dependence of frequency and dwell-time. Notably, the current blockade frequency as well as the current blockade duration increase with increasing the magnitude of applied voltage. Our results suggest that CBn behave as positively charged molecular containers and trans negative voltage promotes their blocking the Ael pore lumen for up to several seconds. Our approach could be used for rapid and real-time probing of the abilities of different CBn to form host-guest complexes with various guest molecules.