Nanopore Analysis of Wild-Type and Mutant Prion Protein (PrPC): Single Molecule Discrimination and PrPC Kinetics

Prion diseases are fatal neurodegenerative diseases associated with the conversion of cellular prion protein (PrP^C) in the central nervous system into the infectious isoform (PrP^Sc). The mechanics of conversion are almost entirely unknown, with understanding stymied by the lack of an atomic-level structure for PrP^Sc. A number of pathogenic PrP^C mutants exist that are characterized by an increased propensity for conversion into PrP^Sc and that differ from wild-type by only a single amino-acid point mutation in their primary structure. These mutations are known to perturb the stability and conformational dynamics of the protein. Understanding of how this occurs may provide insight into the mechanism of PrP^C conversion. In this work we sought to explore wild-type and pathogenic mutant prion protein structure and dynamics by analysis of the current fluctuations through an organic α-hemolysin nanometer-scale pore (nanopore) in which a single prion protein has been captured electrophoretically. In doing this, we find that wild-type and D178N mutant PrP^C, (a PrP^C mutant associated with both Fatal Familial Insomnia and Creutzfeldt-Jakob disease), exhibit easily distinguishable current signatures and kinetics inside the pore and we further demonstrate, with the use of Hidden Markov Model signal processing, accurate discrimination between these two proteins at the single molecule level based on the kinetics of a single PrP^C capture event. Moreover, we present a four-state model to describe wild-type PrP^C kinetics in the pore as a first step in our investigation on characterizing the differences in kinetics and conformational dynamics between wild-type and D178N mutant PrP^C. These results demonstrate the potential of nanopore analysis for highly sensitive, real-time protein and small molecule detection based on single molecule kinetics inside a nanopore, and show the utility of this technique as an assay to probe differences in stability between wild-type and mutant prion proteins at the single molecule level.     

A Brownian ratchet for protein translocation including dissociation of ratcheting sites

We study a model for the translocation of proteins across membranes through a nanopore using a ratcheting mechanism. When the protein enters the nanopore it diffuses in and out of the pore according to a Brownian motion. Moreover, it is bound by ratcheting molecules which hinder the diffusion of the protein out of the nanopore, i.e. the Brownian motion is reflected such that no ratcheting molecule exits the pore. New ratcheting molecules bind at rate γ. Extending our previous approach (Depperschmidt and Pfaffelhuber in Stoch Processes Appl 120:901–925, 2010) we allow the ratcheting molecules to dissociate (at rate δ) from the protein (Model I). We also provide an approximate model (Model II) which assumes a Poisson equilibrium of ratcheting molecules on one side of the current reflection boundary. Using analytical methods and simulations we show that the speeds of both models are approximately the same. Our analytical results on Model II give the speed of translocation by means of a solution of an ordinary differential equation. This speed gives an approximation for the time it takes to translocate a protein of given length.     

Microscopic Mechanics of Hairpin DNA Translocation through Synthetic Nanopores

Nanoscale pores have proved useful as a means to assay DNA and are actively being developed as the basis of genome sequencing methods. Hairpin DNA (hpDNA), having both double-helical and overhanging coil portions, can be trapped in a nanopore, giving ample time to execute a sequence measurement. In this article, we provide a detailed account of hpDNA interaction with a synthetic nanopore obtained through extensive all-atom molecular dynamics simulations. For synthetic pores with minimum diameters from 1.3 to 2.2 nm, we find that hpDNA can translocate by three modes: unzipping of the double helix and—in two distinct orientations—stretching/distortion of the double helix. Furthermore, each of these modes can be selected by an appropriate choice of the pore size and voltage applied transverse to the membrane. We demonstrate that the presence of hpDNA can dramatically alter the distribution of ions within the pore, substantially affecting the ionic current through it. In experiments and simulations, the ionic current relative to that in the absence of DNA can drop below 10% and rise beyond 200%. Simulations associate the former with the double helix occupying the constriction and the latter with accumulation of DNA that has passed through the constriction.     

How Nanopore Translocation Experiments Can Measure RNA Unfolding

Electrokinetic translocation of biomolecules through solid-state nanopores represents a label-free single-molecule technique that may be used to measure biomolecular structure and dynamics. Recent investigations have attempted to distinguish individual transfer RNA (tRNA) species based on the associated pore translocation times, ion-current noise, and blockage currents. By manufacturing sufficiently smaller pores, each tRNA is required to undergo a deformation to translocate. Accordingly, differences in nanopore translocation times and distributions may be used to infer the mechanical properties of individual tRNA molecules. To bridge our understanding of tRNA structural dynamics and nanopore measurements, we apply molecular dynamics simulations using a simplified “structure-based” energetic model. Calculating the free-energy landscape for distinct tRNA species implicates transient unfolding of the terminal RNA helix during nanopore translocation. This provides a structural and energetic framework for interpreting current experiments, which can aid the design of methods for identifying macromolecules using nanopores.     

Nucleotide discrimination with DNA immobilized in the MspA nanopore

Nanopore sequencing has the potential to become a fast and low-cost DNA sequencing platform. An ionic current passing through a small pore would directly map the sequence of single stranded DNA (ssDNA) driven through the constriction. The pore protein, MspA, derived from Mycobacterium smegmatis, has a short and narrow channel constriction ideally suited for nanopore sequencing. To study MspA's ability to resolve nucleotides, we held ssDNA within the pore using a biotin-NeutrAvidin complex. We show that homopolymers of adenine, cytosine, thymine, and guanine in MspA exhibit much larger current differences than in α-hemolysin. Additionally, methylated cytosine is distinguishable from unmethylated cytosine. We establish that single nucleotide substitutions within homopolymer ssDNA can be detected when held in MspA's constriction. Using genomic single nucleotide polymorphisms, we demonstrate that single nucleotides within random DNA can be identified. Our results indicate that MspA has high signal-to-noise ratio and the single nucleotide sensitivity desired for nanopore sequencing devices.     

Microscopic Kinetics of DNA Translocation through synthetic nanopores

We have previously demonstrated that a nanometer-diameter pore in a nanometer-thick metal-oxide-semiconductor-compatible membrane can be used as a molecular sensor for detecting DNA. The prospects for using this type of device for sequencing DNA are avidly being pursued. The key attribute of the sensor is the electric field-induced (voltage-driven) translocation of the DNA molecule in an electrolytic solution across the membrane through the nanopore. To complement ongoing experimental studies developing such pores and measuring signals in response to the presence of DNA, we conducted molecular dynamics simulations of DNA translocation through the nanopore. A typical simulated system included a patch of a silicon nitride membrane dividing water solution of potassium chloride into two compartments connected by the nanopore. External electrical fields induced capturing of the DNA molecules by the pore from the solution and subsequent translocation. Molecular dynamics simulations suggest that 20-basepair segments of double-stranded DNA can transit a nanopore of 2.2 x 2.6 nm(2) cross section in a few microseconds at typical electrical fields. Hydrophobic interactions between DNA bases and the pore surface can slow down translocation of single-stranded DNA and might favor unzipping of double-stranded DNA inside the pore. DNA occluding the pore mouth blocks the electrolytic current through the pore; these current blockades were found to have the same magnitude as the blockade observed when DNA transits the pore. The feasibility of using molecular dynamics simulations to relate the level of the blocked ionic current to the sequence of DNA was investigated.     

Control of subunit stoichiometry in single-chain MspA nanopores

Transmembrane protein channels enable fast and highly sensitive detection of single molecules. Nanopore sequencing of DNA was achieved using an engineered Mycobacterium smegmatis porin A (MspA) in combination with a motor enzyme. Due to its favorable channel geometry, the octameric MspA pore exhibits the highest current level compared with other pore proteins. To date, MspA is the only protein nanopore with a published record of DNA sequencing. While widely used in commercial devices, nanopore sequencing of DNA suffers from significant base-calling errors due to stochastic events of the complex DNA-motor-pore combination and the contribution of up to five nucleotides to the signal at each position. Different mutations in specific subunits of a pore protein offer an enormous potential to improve nucleotide resolution and sequencing accuracy. However, individual subunits of MspA and other oligomeric protein pores are randomly assembled in vivo and in vitro, preventing the efficient production of designed pores with different subunit mutations. In this study, we converted octameric MspA into a single-chain pore by connecting eight subunits using peptide linkers. Lipid bilayer experiments demonstrated that single-chain MspA formed membrane-spanning channels and discriminated all four nucleotides identical to MspA produced from monomers in DNA hairpin experiments. Single-chain constructs comprising three, five, six, and seven connected subunits assembled to functional channels, demonstrating a remarkable plasticity of MspA to different subunit stoichiometries. Thus, single-chain MspA constitutes a new milestone in the optimization of MspA as a biosensor for DNA sequencing and many other applications by enabling the production of pores with distinct subunit mutations and pore diameters.     

Binding of bovine T194A PrPC by PrPSc-specific antibodies

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases that are based on the misfolding of a cellular prion protein (PrP^C) into an infectious, pathological conformation (PrP^Sc). There is proof-of-principle evidence that a prion vaccine is possible but this is tempered with concerns of the potential dangers associated with induction of immune responses to a widely-expressed self-protein. By targeting epitopes that are specifically exposed upon protein misfolding, our group developed a vaccine that induces PrP^Sc-specific antibody responses. Here we consider the ability of this polyclonal antibody (SN6b) to bind to a mutant of PrP^C associated with spontaneous prion disease. Polyclonal antibodies were selected to mimic the vaccination outcome and also explore all possible protein conformations of the recombinant bovine prion protein with mutation T194A [bPrP(T194A)]. This mutant is a homolog of the human T183A mutation of PrP^C that is associated with early onset of familial dementia. With nanopore analysis, under non-denaturing conditions, we observed binding of the SN6b antibody to bPrP(T194A). This interaction was confirmed through ELISAs as well as immunoprecipitation of the recombinant and cellularly expressed forms of bPrP(T194A). This interaction did not promote formation of a protease resistant conformation of PrP in vitro. Collectively, these findings support the disease-specific approach for immunotherapy of prion diseases but also suggest that the concept of conformation-specific immunotherapy may be complicated in individuals who are genetically predisposed to PrP^C misfolding.     

A parallel graph decomposition algorithm for DNA sequencing with nanopores

MOTIVATION: With the potential availability of nanopore devices that can sense the bases of translocating single-stranded DNA (ssDNA), it is likely that 'reads' of length approximately 10(5) will be available in large numbers and at high speed. We address the problem of complete DNA sequencing using such reads.We assume that approximately 10(2) copies of a DNA sequence are split into single strands that break into randomly sized pieces as they translocate the nanopore in arbitrary orientations. The nanopore senses and reports each individual base that passes through, but all information about orientation and complementarity of the ssDNA subsequences is lost. Random errors (both biological and transduction) in the reads create further complications. RESULTS: We have developed an algorithm that addresses these issues. It can be considered an extreme variation of the well-known Eulerian path approach. It searches over a space of de Bruijn graphs until it finds one in which (a) the impact of errors is eliminated and (b) both possible orientations of the two ssDNA sequences can be identified separately and unambiguously.Our algorithm is able to correctly reconstruct real DNA sequences of the order of 10(6) bases (e.g. the bacterium Mycoplasma pneumoniae) from simulated erroneous reads on a modest workstation in about 1 h. We describe, and give measured timings of, a parallel implementation of this algorithm on the Cray Multithreaded Architecture (MTA-2) supercomputer, whose architecture is ideally suited to this 'unstructured' problem. Our parallel implementation is crucial to the problem of rapidly sequencing long DNA sequences and also to the situation where multiple nanopores are used to obtain a high-bandwidth stream of reads.     

Integration and Oligomerization of Bax Protein in Lipid Bilayers Characterized by Single Molecule Fluorescence Study

Bax is a pro-apoptotic Bcl-2 family protein. The activated Bax translocates to mitochondria, where it forms pore and permeabilizes the mitochondrial outer membrane. This process requires the BH3-only activator protein (i.e. tBid) and can be inhibited by anti-apoptotic Bcl-2 family proteins such as Bcl-x_L. Here by using single molecule fluorescence techniques, we studied the integration and oligomerization of Bax in lipid bilayers. Our study revealed that Bax can bind to lipid membrane spontaneously in the absence of tBid. The Bax pore formation undergoes at least two steps: pre-pore formation and membrane insertion. The activated Bax triggered by tBid or BH3 domain peptide integrates on bilayers and tends to form tetramers, which are termed as pre-pore. Subsequent insertion of the pre-pore into membrane is highly dependent on the composition of cardiolipin in lipid bilayers. Bcl-x_L can translocate Bax from membrane to solution and inhibit the pore formation. The study of Bax integration and oligomerization at the single molecule level provides new evidences that may help elucidate the pore formation of Bax and its regulatory mechanism in apoptosis.     

The potential and challenges of nanopore sequencing

A nanopore-based device provides single-molecule detection and analytical capabilities that are achieved by electrophoretically driving molecules in solution through a nano-scale pore. The nanopore provides a highly confined space within which single nucleic acid polymers can be analyzed at high throughput by one of a variety of means, and the perfect processivity that can be enforced in a narrow pore ensures that the native order of the nucleobases in a polynucleotide is reflected in the sequence of signals that is detected. Kilobase length polymers (single-stranded genomic DNA or RNA) or small molecules (e.g., nucleosides) can be identified and characterized without amplification or labeling, a unique analytical capability that makes inexpensive, rapid DNA sequencing a possibility. Further research and development to overcome current challenges to nanopore identification of each successive nucleotide in a DNA strand offers the prospect of 'third generation' instruments that will sequence a diploid mammalian genome for approximately $1,000 in approximately 24 h.     

Directed evolution of an anti-prion protein scFv fragment to an affinity of 1 pM and its structural interpretation

Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative prion disease affecting cattle that is transmissible to humans, manifesting as a variant of Creutzfeldt-Jakob disease (vCJD) likely following the consumption of meat contaminated with BSE prions. High-affinity antibodies are a prerequisite for the development of simple, highly sensitive and non-invasive diagnostic tests that are able to detect even small amounts of the disease-associated PrP conformer (PrP(Sc)). We describe here the affinity maturation of a single-chain Fv antibody fragment with a binding affinity of 1 pM to a peptide derived from the unstructured region of bovine PrP (BoPrP (90-105)). This is the tightest peptide-binding antibody reported to date and may find useful application in diagnostics, especially when PrP(Sc) is pretreated by denaturation and/or proteolysis for peptide-like presentation. Several rounds of directed evolution and off-rate selection with ribosome display were performed using an antibody library generated from a single PrP binder with error-prone PCR and DNA-shuffling. As the correct determinations of affinities in this range are not straightforward, competition biosensor techniques and KinExA methods were both applied and compared. Structural interpretation of the affinity improvement was performed based on the crystal structure of the original prion binder in complex with the BoPrP (95-104) peptide by modeling the corresponding mutations.     

Stretching and unzipping nucleic acid hairpins using a synthetic nanopore

We have explored the electromechanical properties of DNA by using an electric field to force single hairpin molecules to translocate through a synthetic pore in a silicon nitride membrane. We observe a threshold voltage for translocation of the hairpin through the pore that depends sensitively on the diameter and the secondary structure of the DNA. The threshold for a diameter 1.5 < d < 2.3 nm is V > 1.5 V, which corresponds to the force required to stretch the stem of the hairpin, according to molecular dynamics simulations. On the other hand, for 1.0 < d < 1.5 nm, the threshold voltage collapses to V < 0.5 V because the stem unzips with a lower force than required for stretching. The data indicate that a synthetic nanopore can be used like a molecular gate to discriminate between the secondary structures in DNA.     

An ON/OFF biosensor based on blockade of ionic current passing through a solid-state nanopore

Single nanopores have attracted interest for their use as biosensing devices. In general, methods involve measuring ionic current blockades associated with translocation of analytes through the nanopore, but the detection of such short time lasting events requires complex equipment and setup that are critical for convenient routine biosensing. Here we present a novel biosensing concept based on a single nanopore in a silicon nitride membrane and two anchor-linked DNA species that forms trans-pore hybrids, realizing a stable blockade of ionic current through the pore. Molecular recognition events affecting the DNA hybrids cause a pore opening and the consequent establishment of an ionic current. In the present implementation of the device, we constructed a magnetic bead/streptavidin/biotin-DNA1/DNA2-biotin/streptavidin/Quantumdot-cluster complex (where DNA1 is a mismatched reverse complement of DNA2) through a sub-micrometric pore and monitored DNA strand displacement events occurring after addition of an oligonucleotide complementary to DNA2. The electric and mechanical aspects of the novel device, as well as its potential in biosensing are discussed.     

Cytosolic prion protein (PrP) is not toxic in N2a cells and primary neurons expressing pathogenic PrP mutations

Inherited prion diseases are linked to mutations in the prion protein (PrP) gene, which favor conversion of PrP into a conformationally altered, pathogenic isoform. The cellular mechanism by which this process causes neurological dysfunction is unknown. It has been proposed that neuronal death can be triggered by accumulation of PrP in the cytosol because of impairment of proteasomal degradation of misfolded PrP molecules retrotranslocated from the endoplasmic reticulum (Ma, J., Wollmann, R., and Lindquist, S. (2002) Science 298, 1781-1785). To test whether this neurotoxic mechanism is operative in inherited prion diseases, we evaluated the effect of proteasome inhibitors on the viability of transfected N2a cells and primary neurons expressing mouse PrP homologues of the D178N and nine octapeptide mutations. We found that the inhibitors caused accumulation of an unglycosylated, aggregated form of PrP exclusively in transfected N2a expressing PrP from the cytomegalovirus promoter. This form contained an uncleaved signal peptide, indicating that it represented polypeptide chains that had failed to translocate into the ER lumen during synthesis, rather than retrogradely translocated PrP. Quantification of N2a viability in the presence of proteasome inhibitors demonstrated that accumulation of this form was not toxic. No evidence of cytosolic PrP was found in cerebellar granule neurons from transgenic mice expressing wild-type or mutant PrPs from the endogenous promoter, nor were these neurons more susceptible to proteasome inhibitor toxicity than neurons from PrP knock-out mice. Our analysis fails to confirm the previous observation that mislocation of PrP in the cytosol is neurotoxic, and argues against the hypothesis that perturbation of PrP metabolism through the proteasomal pathway plays a pathogenic role in prion diseases.     

Microsecond time-scale discrimination among polycytidylic acid, polyadenylic acid, and polyuridylic acid as homopolymers or as segments within single RNA molecules

Single molecules of DNA or RNA can be detected as they are driven through an alpha-hemolysin channel by an applied electric field. During translocation, nucleotides within the polynucleotide must pass through the channel pore in sequential, single-file order because the limiting diameter of the pore can accommodate only one strand of DNA or RNA at a time. Here we demonstrate that this nanopore behaves as a detector that can rapidly discriminate between pyrimidine and purine segments along an RNA molecule. Nanopore detection and characterization of single molecules represent a new method for directly reading information encoded in linear polymers, and are critical first steps toward direct sequencing of individual DNA and RNA molecules.     

Mutational analysis of topological determinants in prion protein (PrP) and measurement of transmembrane and cytosolic PrP during prion infection

The prion protein (PrP) can adopt multiple membrane topologies, including a fully translocated form (SecPrP), two transmembrane forms (NtmPrP and CtmPrP), and a cytosolic form. It is important to understand the factors that influence production of these species, because two of them, CtmPrP and cytosolic PrP, have been proposed to be key neurotoxic intermediates in certain prion diseases. In this paper, we perform a mutational analysis of PrP synthesized using an in vitro translation system in order to further define sequence elements that influence the formation of CtmPrP. We find that substitution of charged residues in the hydrophobic core of the signal peptide increases synthesis of CtmPrP and also reduces the efficiency of translocation into microsomes. Combining these mutations with substitutions in the transmembrane domain causes the protein to be synthesized exclusively with the CtmPrP topology. Reducing the spacing between the signal peptide and the transmembrane domain also increases CtmPrP. In contrast, topology is not altered by mutations that prevent signal peptide cleavage or by deletion of the C-terminal signal for glycosylphosphatidylinositol anchor addition. Removal of the signal peptide completely blocks translocation. Taken together, our results are consistent with a model in which the signal peptide and transmembrane domain function in distinct ways as determinants of PrP topology. We also present characterization of an antibody that selectively recognizes CtmPrP and cytosolic PrP by virtue of their uncleaved signal peptides. By using this antibody, as well as the distinctive gel mobility of CtmPrP and cytosolic PrP, we show that the amounts of these two forms in cultured cells and rodent brain are not altered by infection with scrapie prions. We conclude that CtmPrP and cytosolic PrP are unlikely to be obligate neurotoxic intermediates in familial or infectiously acquired prion diseases.     

Transport of alpha-helical peptides through alpha-hemolysin and aerolysin pores

A series of negatively charged alpha-helical peptides of the general formula fluorenylmethoxycarbonyl (Fmoc)-D(x)A(y)K(z) were synthesized, where x and z were 1, 2, or 3 and y was 10, 14, 18, or 22. The translocation of the peptides through single pores, which were self-assembled into lipid membranes, was analyzed by measuring the current blockade i(block) and the duration t(block). The pores were either alpha-hemolysin, which has a wide vestibule leading into the pore, or aerolysin, which has no vestibule but has a longer pore of a similar diameter. Many thousands of events were measured for each peptide with each pore, and they could be assigned to two types: bumping events (type I) have a small i(block) and long t(block), and translocation events (type II) have a larger i(block) and shorter t(block). For type-II events, both i(block) and t(block) increase with the length of the peptides on both pores tested. The dipole moment and the net charge of each peptide has a major effect on the transport characteristics. The ratio of type-II/type-I events increases as the dipole moment increases, and uncharged peptides gave mostly type-I events. The structural differences between the two nanopores were reflected in the characteristic values of i(block), and in particular, the vestibule of alpha-hemolysin helps to orient the peptides for translocation. Overall, the results demonstrate that the nanopore technology can provide useful structural information but peptide sequencing will require further improvements in the design of the pores.     

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.     

Nanopore unitary permeability measured by electrochemical and optical single transporter recording

For the analysis of membrane transport processes two single molecule methods are available that differ profoundly in data acquisition principle, achievable information, and application range: the widely employed electrical single channel recording and the more recently established optical single transporter recording. In this study dense arrays of microscopic horizontal bilayer membranes between 0.8 microm and 50 microm in diameter were created in transparent foils containing either microholes or microcavities. Prototypic protein nanopores were formed in bilayer membranes by addition of Staphylococcus aureus alpha-hemolysin (alpha-HL). Microhole arrays were used to monitor the formation of bilayer membranes and single alpha-HL pores by confocal microscopy and electrical recording. Microcavity arrays were used to characterize the formation of bilayer membranes and the flux of fluorescent substrates and inorganic ions through single transporters by confocal microscopy. Thus, the unitary permeability of the alpha-HL pore was determined for calcein and Ca(2+) ions. The study paves the way for an amalgamation of electrical and optical single transporter recording. Electro-optical single transporter recording could provide so far unresolved kinetic data of a large number of cellular transporters, leading to an extension of the nanopore sensor approach to the single molecule analysis of peptide transport by translocases.