The emerging landscape of single-molecule protein sequencing technologies.

Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics.

Seeing the Invisibles: Detection of Peptide Enantiomers, Diastereomers, and Isobaric Ring Formation in Lanthipeptides Using Nanopores.

Mass spectrometry (MS) is widely used in proteomic analysis but cannot differentiate between molecules with the same mass-to-charge ratio. Nanopore technology might provide an alternative method for the rapid and cost-effective analysis and sequencing of proteins. In this study, we demonstrate that nanopore currents can distinguish between diastereomeric and enantiomeric differences in l- and d-peptides, not observed by conventional MS analysis, down to individual d-amino acids in small opioid peptides. Molecular dynamics simulations suggest that similar to chiral chromatography the resolution likely arises from multiple chiral interactions during peptide transport across the nanopore. Additionally, we used nanopore recordings to rapidly assess 4- and 11-amino acid ring formation in lanthipeptides, a process used in the synthesis of pharmaceutical peptides. The cyclization step requires distinguishing between constitutional isomers, which have identical MS signals and typically involve numerous tedious experiments to confirm. Hence, nanopore technology offers new possibilities for the rapid and cost-effective analysis of peptides, including those that cannot be easily differentiated by mass spectrometry.

Quantification of Protein Glycosylation Using Nanopores.

Although nanopores can be used for single-molecule sequencing of nucleic acids using low-cost portable devices, the characterization of proteins and their modifications has yet to be established. Here, we show that hydrophilic or glycosylated peptides translocate too quickly across FraC nanopores to be recognized. However, high ionic strengths (i.e., 3 M LiCl) and low pH (i.e., pH 3) together with using a nanopore with a phenylalanine at its constriction allows the recognition of hydrophilic peptides, and to distinguish between mono- and diglycosylated peptides. Using these conditions, we devise a nanopore method to detect, characterize, and quantify post-translational modifications in generic proteins, which is one of the pressing challenges in proteomic analysis.

Autonomous and Active Transport Operated by an Entropic DNA Piston.

We present a synthetic nanoscale piston that uses chemical energy to perform molecular transport against an applied bias. Such a device comprises a 13 by 5 nm protein cylinder, embedded in a biological membrane enclosing a single-stranded DNA (ssDNA) rod. Hybridization with DNA cargo rigidifies the rod, allowing for transport of a selected DNA molecule across the nanopore. A strand displacement reaction from ssDNA fuel on the other side of the membrane then liberates the DNA cargo back into solution and regenerates the initial configuration. The entropic penalty of ssDNA confinement inside the nanopore drives DNA transport regardless of the applied bias. Multiple automated and reciprocating cycles are observed, in which the DNA piston moves through the 10 nm length of the nanopore. In every cycle, a single DNA molecule is transported across the nanopore against an external bias force, which is the hallmark of biological transporters.

PlyAB Nanopores Detect Single Amino Acid Differences in Folded Haemoglobin from Blood.

The real-time identification of protein biomarkers is crucial for the development of point-of-care and portable devices. Here, we use a PlyAB biological nanopore to detect haemoglobin (Hb) variants. Adult haemoglobin (HbA) and sickle cell anaemia haemoglobin (HbS), which differ by just one amino acid, were distinguished in a mixture with more than 97 % accuracy based on individual blockades. Foetal Hb, which shows a larger sequence variation, was distinguished with near 100 % accuracy. Continuum and Brownian dynamics simulations revealed that Hb occupies two energy minima, one near the inner constriction and one at the trans entry of the nanopore. Thermal fluctuations, the charge of the protein, and the external bias influence the dynamics of Hb within the nanopore, which in turn generates the unique ionic current signal in the Hb variants. Finally, Hb was counted from blood samples, demonstrating that direct discrimination and quantification of Hb from blood using nanopores, is feasible.

Electro-Osmotic Vortices Promote the Capture of Folded Proteins by PlyAB Nanopores.

Biological nanopores are emerging as powerful tools for single-molecule analysis and sequencing. Here, we engineered the two-component pleurotolysin (PlyAB) toxin to assemble into 7.2 × 10.5 nm cylindrical nanopores with a low level of electrical noise in lipid bilayers, and we addressed the nanofluidic properties of the nanopore by continuum simulations. Surprisingly, proteins such as human albumin (66.5 kDa) and human transferrin (76-81 kDa) did not enter the nanopore. We found that the precise engineering of the inner surface charge of the PlyAB induced electro-osmotic vortices that allowed the electrophoretic capture of the proteins. Once inside the nanopore, two human plasma proteins could be distinguished by the characteristics of their current blockades. This fundamental understanding of the nanofluidic properties of nanopores provides a practical method to promote the capture and analysis of folded proteins by nanopores.

Automated Electrical Quantification of Vitamin†B1 in a Bodily Fluid using an Engineered Nanopore Sensor.

The ability to measure the concentration of metabolites in biological samples is important, both in the clinic and for home diagnostics. Here we present a nanopore-based biosensor and automated data analysis for quantification of thiamine in urine in less than a minute, without the need for recalibration. For this we use the Cytolysin A nanopore and equip it with an engineered periplasmic thiamine binding protein (TbpA). To allow fast measurements we tuned the affinity of TbpA for thiamine by redesigning the π-π stacking interactions between the thiazole group of thiamine and TbpA. This substitution resulted furthermore in a marked difference between unbound and bound state, allowing the reliable discrimination of thiamine from its two phosphorylated forms by residual current only. Using an array of nanopores, this will allow the quantification within seconds, paving the way for next-generation single-molecule metabolite detection systems.

Label-Free Detection of Post-translational Modifications with a Nanopore.

Post-translational modifications (PTMs) of proteins play key roles in cellular processes. Hence, PTM identification is crucial for elucidating the mechanism of complex cellular processes and disease. Here we present a method for PTM detection at the single-molecule level using FraC biological nanopores. We focus on two major PTMs, phosphorylation and glycosylation, that mutually compete for protein modification sites, an important regulatory process that has been implicated in the pathogenic pathways of many diseases. We show that phosphorylated and glycosylated peptides can be clearly differentiated from nonmodified peptides by differences in the relative current blockade and dwell time in nanopore translocations. Furthermore, we show that these PTM modifications can be mutually differentiated, demonstrating the identification of phosphorylation and glycosylation in a label-free manner. The results represent an important step for the single-molecule, label-free identification of proteoforms, which have tremendous potential for disease diagnosis and cell biology.

Reversible Photocontrolled Nanopore Assembly.

Self-assembly is a fundamental feature of biological systems, and control of such processes offers fascinating opportunities to regulate function. Fragaceatoxin C (FraC) is a toxin that upon binding to the surface of sphingomyelin-rich cells undergoes a structural metamorphosis, leading to the assembly of nanopores at the cell membrane and causing cell death. In this study we attached photoswitchable azobenzene pendants to various locations near the sphingomyelin binding pocket of FraC with the aim of remote controlling the nanopore assembly using light. We found several constructs in which the affinity of the toxin for biological membranes could be activated or deactivated by irradiation, thus enabling reversible photocontrol of pore formation. Notably, one construct was completely inactive in the thermally adapted state; it however induced full lysis of cultured cancer cells upon light irradiation. Selective irradiation also allowed isolation of individual nanopores in artificial lipid membranes. Photocontrolled FraC might find applications in photopharmacology for cancer therapeutics and has potential to be used for the fabrication of nanopore arrays in nanopore sensing devices, where the reconstitution, with high spatiotemporal precision, of single nanopores must be controlled.

Aß42 assembles into specific ß-barrel pore-forming oligomers in membrane-mimicking environments.

The formation of amyloid-ß peptide (Aß) oligomers at the cellular membrane is considered to be a crucial process underlying neurotoxicity in Alzheimer's disease (AD). Therefore, it is critical to characterize the oligomers that form within a membrane environment. To contribute to this characterization, we have applied strategies widely used to examine the structure of membrane proteins to study the two major Aß variants, Aß40 and Aß42. Accordingly, various types of detergent micelles were extensively screened to identify one that preserved the properties of Aß in lipid environments-namely the formation of oligomers that function as pores. Remarkably, under the optimized detergent micelle conditions, Aß40 and Aß42 showed different behavior. Aß40 aggregated into amyloid fibrils, whereas Aß42 assembled into oligomers that inserted into lipid bilayers as well-defined pores and adopted a specific structure with characteristics of a ß-barrel arrangement that we named ß-barrel pore-forming Aß42 oligomers (ßPFOsAß42). Because Aß42, relative to Aß40, has a more prominent role in AD, the higher propensity of Aß42 to form ßPFOs constitutes an indication of their relevance in AD. Moreover, because ßPFOsAß42 adopt a specific structure, this property offers an unprecedented opportunity for testing a hypothesis regarding the involvement of ßPFOs and, more generally, membrane-associated Aß oligomers in AD.

Reduction of Folate by Dihydrofolate Reductase from Thermotoga maritima.

Mammalian dihydrofolate reductases (DHFRs) catalyze the reduction of folate more efficiently than the equivalent bacterial enzymes do, despite typically having similar efficiencies for the reduction of their natural substrate, dihydrofolate. In contrast, we show here that DHFR from the hyperthermophilic bacterium Thermotoga maritima can catalyze reduction of folate to tetrahydrofolate with an efficiency similar to that of reduction of dihydrofolate under saturating conditions. Nuclear magnetic resonance and mass spectrometry experiments showed no evidence of the production of free dihydrofolate during either the EcDHFR- or TmDHFR-catalyzed reductions of folate, suggesting that both enzymes perform the two reduction steps without release of the partially reduced substrate. Our results imply that the reaction proceeds more efficiently in TmDHFR than in EcDHFR because the more open active site of TmDHFR facilitates protonation of folate. Because T. maritima lives under extreme conditions where tetrahydrofolate is particularly prone to oxidation, this ability to salvage folate may impart an advantage to the bacterium by minimizing the squandering of a valuable cofactor.

Real-Time Conformational Changes and Controlled Orientation of Native Proteins Inside a Protein Nanoreactor.

Protein conformations play crucial roles in most, if not all, biological processes. Here we show that the current carried through a nanopore by ions allows monitoring conformational changes of single and native substrate-binding domains (SBD) of an ATP-Binding Cassette importer in real-time. Comparison with single-molecule Förster Resonance Energy Transfer and ensemble measurements revealed that proteins trapped inside the nanopore have bulk-like properties. Two ligand-free and two ligand-bound conformations of SBD proteins were inferred and their kinetic constants were determined. Remarkably, internalized proteins aligned with the applied voltage bias, and their orientation could be controlled by the addition of a single charge to the protein surface. Nanopores can thus be used to immobilize proteins on a surface with a specific orientation, and will be employed as nanoreactors for single-molecule studies of native proteins. Moreover, nanopores with internal protein adaptors might find further practical applications in multianalyte sensing devices.

Functional truncated membrane pores.

Membrane proteins are generally divided into two classes. Integral proteins span the lipid bilayer, and peripheral proteins are located at the membrane surface. Here, we provide evidence for membrane proteins of a third class that stabilize lipid pores, most probably as toroidal structures. We examined mutants of the staphylococcal α-hemolysin pore so severely truncated that the protein cannot span a bilayer. Nonetheless, the doughnut-like structures elicited well-defined transmembrane ionic currents by inducing pore formation in the underlying lipids. The formation of lipid pores, produced here by a structurally defined protein, is supported by the lipid and voltage dependences of pore formation, and by molecular dynamics simulations. We discuss the role of stabilized lipid pores in amyloid disease, the action of antimicrobial peptides, and the assembly of the membrane-attack complexes of the immune system.

A Protein Rotaxane Controls the Translocation of Proteins Across a ClyA Nanopore.

Rotaxanes, pseudorotaxanes, and catenanes are supramolecular complexes with potential use in nanomachinery, molecular computing, and single-molecule studies. Here we constructed a protein rotaxane in which a polypeptide thread is encircled by a Cytolysin A (ClyA) nanopore and capped by two protein stoppers. The rotaxane could be switched between two states. At low negative applied potentials (<-50 mV) one of the protein stoppers resided inside the nanopore indefinitely. Under this configuration the rotaxane prevents the diffusion of protein molecules across the lipid bilayer and provides a useful platform for single-molecule analysis. High negative applied potentials (-100 mV) dismantled the interlocked rotaxane system by the forceful translocation of the protein stopper, allowing new proteins to be trapped inside or transported across the nanopore. The observed voltage threshold for the translocation of the protein stopper through the nanopore related well to the biphasic voltage dependence of the residence time measured for the freely diffusing protein stopper. We propose a model in which molecules translocate through a nanopore when the average dwell time decreases with the applied potential.

Alpha-Helical Fragaceatoxin†C Nanopore Engineered for Double-Stranded and Single-Stranded Nucleic Acid Analysis.

Nanopores are used in single-molecule DNA analysis and sequencing. Herein, we show that Fragaceatoxin C (FraC), an α-helical pore-forming toxin from an actinoporin protein family, can be reconstituted in sphingomyelin-free standard planar lipid bilayers. We engineered FraC for DNA analysis and show that the funnel-shaped geometry allows tight wrapping around single-stranded DNA (ssDNA), resolving between homopolymeric C, T, and A polynucleotide stretches. Remarkably, despite the 1.2 nm internal constriction of FraC, double-stranded DNA (dsDNA) can translocate through the nanopore at high applied potentials, presumably through the deformation of the α-helical transmembrane region of the pore. Therefore, FraC nanopores might be used in DNA sequencing and dsDNA analysis.

Single-Molecule Analyte Recognition with ClyA Nanopores Equipped with Internal Protein Adaptors.

Nanopores have been used to detect molecules, to sequence DNA, or to investigate chemical reactions at the single-molecule level. Because they approach the absolute limit of sensor miniaturization, nanopores are amenable to parallelization and could be used in single-cell measurements. Here we show that single enzymes can be functionally and reversibly trapped inside the confined space of a ClyA nanopore. Remarkably, the binding of ligands to the internalized proteins is mirrored by specific changes to the nanopore conductance. Conveniently, the manipulation of the charge of the protein allowed increasing of the residence time of the protein inside the nanopore. Nanopores with internalized protein adaptors can be used to study proteins in real time or can be incorporated into inexpensive portable devices for the detection of analytes with high selectivity.

DNA stretching and optimization of nucleobase recognition in enzymatic nanopore sequencing.

In nanopore sequencing, where single DNA strands are electrophoretically translocated through a nanopore and the resulting ionic signal is used to identify the four DNA bases, an enzyme has been used to ratchet the nucleic acid stepwise through the pore at a controlled speed. In this work, we investigated the ability of alpha-hemolysin nanopores to distinguish the four DNA bases under conditions that are compatible with the activity of DNA-handling enzymes. Our findings suggest that in immobilized strands, the applied potential exerts a force on DNA (∼10 pN at +160 mV) that increases the distance between nucleobases by about 2.2 ŠV(-1). The four nucleobases can be resolved over wide ranges of applied potentials (from +60 to +220 mV in 1 m KCl) and ionic strengths (from 200 mM KCl to 1 M KCl at +160 mV) and nucleobase recognition can be improved when the ionic strength on the side of the DNA-handling enzyme is low, while the ionic strength on the opposite side is high.

Unravelled proteins form blobs during translocation across nanopores.

The electroosmotic-driven transport of unravelled proteins across nanopores is an important biological process that is now under investigation for the rapid analysis and sequencing of proteins. For this approach to work, however, it is crucial that the polymer is threaded in single file. Here we found that, contrary to the electrophoretic transport of charged polymers such as DNA, during polypeptide translocation blob-like structures typically form inside nanopores. Comparisons between different nanopore sizes, shapes and surface chemistries showed that under electroosmotic-dominated regimes single-file transport of polypeptides can be achieved using nanopores that simultaneously have an entry and an internal diameter that is smaller than the persistence length of the polymer, have a uniform non-sticky ( i . e . non-aromatic) nanopore inner surface, and using moderate translocation velocities.

Visible Light Control over the Cytolytic Activity of a Toxic Pore-Forming Protein.

Enabling control over the bioactivity of proteins with light, along with the principles of photopharmacology, has the potential to generate safe and targeted medical treatments. Installing light sensitivity in a protein can be achieved through its covalent modification with a molecular photoswitch. The general challenge in this approach is the need for the use of low energy visible light for the regulation of bioactivity. In this study, we report visible light control over the cytolytic activity of a protein. A water-soluble visible-light-operated tetra-ortho-fluoro-azobenzene photoswitch was synthesized by utilizing the nucleophilic aromatic substitution reaction for installing a solubilizing sulfonate group onto the electron-poor photoswitch structure. The azobenzene was attached to two cysteine mutants of the pore-forming protein fragaceatoxin C (FraC), and their respective activities were evaluated on red blood cells. For both mutants, the green-light-irradiated sample, containing predominantly the cis-azobenzene isomer, was more active compared to the blue-light-irradiated sample. Ultimately, the same modulation of the cytolytic activity pattern was observed toward a hypopharyngeal squamous cell carcinoma. These results constitute the first case of using low energy visible light to control the biological activity of a toxic protein.