Discrimination between Alpha-Synuclein Protein Variants with a Single Nanometer-Scale Pore.

Alpha-synuclein is one of several key factors in the regulation of nerve activity. It is striking that single- or multiple-point mutations in the 140-amino-acid-long protein can change its structure, which leads to the protein's aggregation and fibril formation (which is associated with several neurodegenerative diseases, e.g., Parkinson's disease). We recently demonstrated that a single nanometer-scale pore can identify proteins based on its ability to discriminate between protease-generated polypeptide fragments. We show here that a variation of this method can readily discriminate between the wild-type alpha synuclein, a known deleterious point mutation of the glutamic acid at position 46 replaced with a lysine (E46K), and post-translational modifications (i.e., tyrosine Y39 nitration and serine 129 phosphorylation).

Comprehensive structural assignment of glycosaminoglycan oligo- and polysaccharides by protein nanopore.

Glycosaminoglycans are highly anionic functional polysaccharides with information content in their structure that plays a major role in the communication between the cell and the extracellular environment. The study presented here reports the label-free detection and analysis of glycosaminoglycan molecules at the single molecule level using sensing by biological nanopore, thus addressing the need to decipher structural information in oligo- and polysaccharide sequences, which remains a major challenge for glycoscience. We demonstrate that a wild-type aerolysin nanopore can detect and characterize glycosaminoglycan oligosaccharides with various sulfate patterns, osidic bonds and epimers of uronic acid residues. Size discrimination of tetra- to icosasaccharides from heparin, chondroitin sulfate and dermatan sulfate was investigated and we show that different contents and distributions of sulfate groups can be detected. Remarkably, differences in α/ß anomerization and 1,4/1,3 osidic linkages can also be detected in heparosan and hyaluronic acid, as well as the subtle difference between the glucuronic/iduronic epimers in chondroitin and dermatan sulfate. Although, at this stage, discrimination of each of the constituent units of GAGs is not yet achieved at the single-molecule level, the resolution reached in this study is an essential step toward this ultimate goal.

On possible trypsin-induced biases in peptides analysis with aerolysin nanopore.

Nanopore-based single-molecule analysis technique is a promising approach in the field of proteomics. In this Technical Brief, the interaction between the biological nanopore of Aerolysin (AeL) and peptides is investigated, focusing on potential biases depending on the AeL activation protocol. Our results reveal that residual trypsin, which may be unintentionally introduced in analyte solution when using a classical AeL activation protocol, can induce a significant formation of shorter peptides by enzymatic degradation of longer ones, which may lead to unwanted effects and/or misinterpretations. AeL free-trypsin activation protocol eliminates this bias and appears more appropriate for peptide/proteins analysis, specifically in the perspective of nanopore-based molecular fingerprinting or of low-abundance species characterization.

Nanopore-Based Protein Identification.

The implementation of a reliable, rapid, inexpensive, and simple method for whole-proteome identification would greatly benefit cell biology research and clinical medicine. Proteins are currently identified by cleaving them with proteases, detecting the polypeptide fragments with mass spectrometry, and mapping the latter to sequences in genomic/proteomic databases. Here, we demonstrate that the polypeptide fragments can instead be detected and classified at the single-molecule limit using a nanometer-scale pore formed by the protein aerolysin. Specifically, three different water-soluble proteins treated with the same protease, trypsin, produce different polypeptide fragments defined by the degree by which the latter reduce the nanopore's ionic current. The fragments identified with the aerolysin nanopore are consistent with the predicted fragments that trypsin could produce.

Pore-forming toxins as tools for polymer analytics: From sizing to sequencing.

We report here on the nanopore resistive pulse sensing (Np-RPS) method, involving pore-forming toxins as tools for polymer analytics at single molecule level. Np-RPS is an electrical method for the label-free detection of single molecules. A molecule interacting with the pore causes a change of the electrical resistance of the pore, called a resistive pulse, associated with a measurable transient current blockade. The features of the blockades, in particular their depth and duration, contain information on the molecular properties of the analyte. We first revisit the history of Np-RPS, then we discuss the effect of the configuration of the molecule/nanopore interaction on the molecular information that can be extracted from the signal, illustrated in two different regimes that either favor molecular sequencing or molecular sizing. Specifically, we focus on the sizing regime and on the use of two different pore-forming toxins, staphylococcal α-hemolysin (αHL) and aerolysin (AeL) nanopores, for the characterization of water-soluble polymers (poly-(ethylene glycol), (PEG)), homopeptides, and heteropeptides. We discuss how nanopore sizing of polymers could be envisioned as a new approach for peptide/protein sequencing.

Aerolysin, a Powerful Protein Sensor for Fundamental Studies and Development of Upcoming Applications.

The nanopore electrical approach is a breakthrough in single molecular level detection of particles as small as ions, and complex as biomolecules. This technique can be used for molecule analysis and characterization as well as for the understanding of confined medium dynamics in chemical or biological reactions. Altogether, the information obtained from these kinds of experiments will allow us to address challenges in a variety of biological fields. The sensing, design, and manufacture of nanopores is crucial to realize these objectives. For some time now, aerolysin, a pore forming toxin, and its mutants have shown high potential in real time analytical chemistry, size discrimination of neutral polymers, oligosaccharides, oligonucleotides and peptides at monomeric resolution, sequence identification, chemical modification on DNA, potential biomarkers detection, and protein folding analysis. This review focuses on the results obtained with aerolysin nanopores on the fields of chemistry, biology, physics, and biotechnology. We discuss and compare as well the results obtained with other protein channel sensors.

Dynamics of a polyelectrolyte through aerolysin channel as a function of applied voltage and concentration⋆.

We describe the behaviour of a polyelectrolyte in confined geometry. The transport of a polyelectrolyte, dextran sulfate, through a recombinant protein channel, aerolysin, inserted into a planar lipid bilayer is studied as a function of applied voltage and polyelectrolyte concentration and chain length. The aerolysin pore has a weak geometry asymmetry, a high number of charged residues and the polyelectrolyte is strongly negatively charged. The resulting current blockades were characterized by short and long dwelling times. Their frequency varies exponentially as a function of applied voltage and linearly as a function of polyelectrolyte concentration. The long blockade duration decreases exponentially when the electrical force increases. The ratio of the population of short events to the one of long events decreases when the applied voltage increases and displays an exponential variation. The long residence time increases with the polyelectrolyte chain length. We measure a reduction of the effective charge of the polyelectrolyte at the pore entry and inside the channel. For a fixed applied voltage, + / - 100 mV, at both sides of the protein pore entrance, the events frequency is similar as a function of dextran sulfate concentration. The mean blockade durations are independent of polyelectrolyte concentration and are similar for both entrances of the pore and remain constant as a function of the electrical force.

Identification of single amino acid differences in uniformly charged homopolymeric peptides with aerolysin nanopore.

There are still unmet needs in finding new technologies for biomedical diagnostic and industrial applications. A technology allowing the analysis of size and sequence of short peptide molecules of only few molecular copies is still challenging. The fast, low-cost and label-free single-molecule nanopore technology could be an alternative for addressing these critical issues. Here, we demonstrate that the wild-type aerolysin nanopore enables the size-discrimination of several short uniformly charged homopeptides, mixed in solution, with a single amino acid resolution. Our system is very sensitive, allowing detecting and characterizing a few dozens of peptide impurities in a high purity commercial peptide sample, while conventional analysis techniques fail to do so.

Probing driving forces in aerolysin and α-hemolysin biological nanopores: electrophoresis versus electroosmosis.

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.

Temperature Effect on Ionic Current and ssDNA Transport through Nanopores.

We have investigated the role of electrostatic interactions in the transport of nucleic acids and ions through nanopores. The passage of DNA through nanopores has so far been conjectured to involve a free-energy barrier for entry, followed by a downhill translocation where the driving voltage accelerates the polymer. We have tested the validity of this conjecture by using two toxins, α-hemolysin and aerolysin, which differ in their shape, size, and charge. The characteristic timescales in each toxin as a function of temperature show that the entry barrier is ∼15 kBT and the translocation barrier is ∼35 kBT, although the electrical force in the latter step is much stronger. Resolution of this fact, using a theoretical model, reveals that the attraction between DNA and the charges inside the barrel of the pore is the most dominant factor in determining the translocation speed and not merely the driving electrochemical potential gradient.

Evidence of unfolded protein translocation through a protein nanopore.

Protein nanopores are mainly used to study transport, unfolding, intrinsically disordered proteins, protein-pore interactions, and protein-ligand complexes. This single-molecule sensor for biomedical and biotechnological applications is promising but until now direct proof of protein translocation through a narrow channel is lacking. Here, we report the translocation of a chimera molecule through the aerolysin nanopore in the presence of a denaturing agent, guanidium chloride (1.5 M) and KCl (1 M). The chimera molecule is composed of the recombinant MalE protein with a unique cysteine residue at the C-terminal position covalently linked to a single-stranded DNA oligonucleotide. Real-time polymerase chain reaction (PCR) was used to detect the presence of chimera molecules that have been effectively translocated from the cis to trans chamber of the set up. Comparing the electrical signature of the chimera related to the protein or oligonucleotide alone demonstrates that each type of molecule displays different dynamics in term of transport time, event frequency, and current blockade. This original approach provides the possibility to study protein translocation through different biological, artificial, and biomimetic nanopores or nanotubes. New future applications are now conceivable such as protein refolding at the nanopore exit, peptides and protein sequencing, and peptide characterization for diagnostics.

Protein unfolding through nanopores.

In this mini-review we introduce and discuss a new method, at single molecule level, to study the protein folding and protein stability, with a nanopore coupled to an electric detection. Proteins unfolded or partially folded passing through one channel submitted to an electric field, in the presence of salt solution, induce different detectable blockades of ionic current. Their duration depends on protein conformation. For different studies proteins through nanopores, completely unfolded proteins induce only short current blockades. Their frequency increases as the concentration of denaturing agent or temperature increases, following a sigmoidal denaturation curve. The geometry or the net charge of the nanopores does not alter the unfolding transition, sigmoidal unfolding curve and half denaturing concentration or half temperature denaturation. A destabilized protein induces a shift of the unfolding curve towards the lower values of the denaturant agent compared to the wild type protein.Partially folded proteins exhibit very long blockades in nanopores. The blockade duration decreases when the concentration of denaturing agent increases. The variation of these blockades could be associated to a possible glassy behaviour.

Kinetics of enzymatic degradation of high molecular weight polysaccharides through a nanopore: experiments and data-modeling.

The enzymatic degradation of long polysaccharide chains is monitored by nanopore detection. It follows a Michaelis-Menten mechanism. We measure the corresponding kinetic constants at the single molecule level. The simulation results of the degradation process allowed one to account for the oligosaccharide size distribution detected by a nanopore.

Sensing proteins through nanopores: fundamental to applications.

Proteins subjected to an electric field and forced to pass through a nanopore induce blockades of ionic current that depend on the protein and nanopore characteristics and interactions between them. Recent advances in the analysis of these blockades have highlighted a variety of phenomena that can be used to study protein translocation and protein folding, to probe single-molecule catalytic reactions in order to obtain kinetic and thermodynamic information, and to detect protein-antibody complexes, proteins with DNA and RNA aptamers, and protein-pore interactions. Nanopore design is now well controlled, allowing the development of future biotechnologies and medicine applications.

Mapping the conformational stability of maltose binding protein at the residue scale using nuclear magnetic resonance hydrogen exchange experiments.

Being able to differentiate local fluctuations from global folding-unfolding dynamics of a protein is of major interest for improving our understanding of structure-function determinants. The maltose binding protein (MBP), a protein that belongs to the maltose transport system, has a structure composed of two globular domains separated by a rigid-body "hinge bending". Here we determined, by using hydrogen exchange (HX) nuclear magnetic resonance experiments, the apparent stabilization free energies of 101 residues of MBP bound to ß-cyclodextrin (MBP-ßCD) under native conditions. We observed that the last helix of MBP (helix α14) has a lower protection factor than the rest of the protein. Further, HX experiments were performed using guanidine hydrochloride under subdenaturing conditions to discriminate between local fluctuations and global unfolding events and to determine the MBP-ßCD energy landscape. The results show that helix α4 and a part of helices α5 and α6 are clearly grouped into a subdenaturing folding unit and represent a partially folded intermediate under native conditions. In addition, we observed that amide protons located in the hinge between the two globular domains share similar ΔG(gu)(app) and m values and should unfold simultaneously. These observations provide new points of view for improving our understanding of the thermodynamic stability and the mechanisms that drive folding-unfolding dynamics of proteins.

Single molecule detection of glycosaminoglycan hyaluronic acid oligosaccharides and depolymerization enzyme activity using a protein nanopore.

Glycosaminoglycans are biologically active anionic carbohydrates that are among the most challenging biopolymers with regards to their structural analysis and functional assessment. The potential of newly introduced biosensors using protein nanopores that have been mainly described for nucleic acids and protein analysis to date, has been here applied to this polysaccharide-based third class of bioactive biopolymer. This nanopore approach has been harnessed in this study to analyze the hyaluronic acid glycosamiglycan and its depolymerization-derived oligosaccharides. The translocation of a glycosaminoglycan is reported using aerolysin protein nanopore. Nanopore translocation of hyaluronic acid oligosaccharides was evidenced by the direct detection of translocated molecules accumulated into the arrival compartment using high-resolution mass spectrometry. Anionic oligosaccharides of various polymerization degrees were discriminated through measurement of the dwelling time and translocation frequency. This molecular sizing capability of the protein nanopore device allowed the real-time recording of the enzymatic cleavage of hyaluronic acid polysaccharide. The time-resolved detection of enzymatically produced oligosaccharides was carried out to monitor the depolymerization enzyme reaction at the single-molecule level.

Protein transport through a narrow solid-state nanopore at high voltage: experiments and theory.

We report experimentally the transport of an unfolded protein through a narrow solid-state nanopore of 3 nm diameter as a function of applied voltage. The random coil polypeptide chain is larger than the nanopore. The event frequency dependency of current blockades from 200 to 750 mV follows a van't Hoff-Arrhenius law due to the confinement of the unfolded chain. The protein is an extended conformation inside the pore at high voltage. We observe that the protein dwell time decreases exponentially at medium voltage and is inversely proportional to voltage for higher values. This is consistent with the translocation mechanism where the protein is confined in the pore, creating an entropic barrier, followed by electrophoretic transport. We compare these results to our previous work with a larger pore of 20 nm diameter. Our data suggest that electro-osmotic flow and protein adsorption on the narrowest nanopore wall are minimized. We discuss the experimental data obtained as compared with recent theory for the polyelectrolyte translocation process. This theory reproduces clearly the experimental crossover between the entropic barrier regime with medium voltage and the electrophoretic regime with higher voltage.

Thermal unfolding of proteins probed at the single molecule level using nanopores.

The nanopore technique has great potential to discriminate conformations of proteins. It is a very interesting system to mimic and understand the process of translocation of biomacromolecules through a cellular membrane. In particular, the unfolding and folding of proteins before and after going through the nanopore are not well understood. We study the thermal unfolding of a protein, probed by two protein nanopores: aerolysin and α-hemolysin. At room temperature, the native folded protein does not enter into the pore. When we increase the temperature from 25 to 50 °C, the molecules unfold and the event frequency of current blockade increases. A similar sigmoid function fits the normalized event frequency evolution for both nanopores, thus the unfolding curve does not depend on the structure and the net charge of the nanopore. We performed also a circular dichroism bulk experiment. We obtain the same melting temperature (around 45 °C) using the bulk and single molecule techniques.

DNA unzipping and protein unfolding using nanopores.

We present here an overview on unfolding of biomolecular structures as DNA double strands or protein folds. After some theoretical considerations giving orders of magnitude about transport timescales through pores, forces involved in unzipping processes … we present our experiments on DNA unzipping or protein unfolding using a nanopore. We point out the difficulties that can be encountered during these experiments, such as the signal analysis problems, noise issues, or experimental limitations of such system.

Wild type, mutant protein unfolding and phase transition detected by single-nanopore recording.

Understanding protein folding remains a challenge. A difficulty is to investigate experimentally all the conformations in the energy landscape. Only single molecule methods, fluorescence and force spectroscopy, allow observing individual molecules along their folding pathway. Here we observe that single-nanopore recording can be used as a new single molecule method to explore the unfolding transition and to examine the conformational space of native or variant proteins. We show that we can distinguish unfolded states from partially folded ones with the aerolysin pore. The unfolding transition curves of the destabilized variant are shifted toward the lower values of the denaturant agent compared to the wild type protein. The dynamics of the partially unfolded wild type protein follows a first-order transition. The denaturation curve obtained with the aerolysin pore is similar to that obtained with the α-hemolysin pore. The nanopore geometry or net charge does not influence the folding transition but changes the dynamics.