Protein nanopores with covalently attached molecular adapters

Molecular adapters are crucial for the stochastic sensing of organic analytes with alpha-hemolysin (alphaHL) protein nanopores when direct interactions between analytes and the pore cannot readily be arranged by conventional protein engineering. In our earlier studies, cyclodextrin adapters were lodged noncovalently within the lumen of the alphaHL pore. In the present work, we have realized the controlled covalent attachment of a beta-cyclodextrin (betaCD) adapter in the two possible molecular orientations inside alphaHL pores prepared by genetic engineering. There are two advantages to such a covalent system. First, the adapter cannot dissociate, which means there are no gaps during stochastic detection, a crucial advance for single-molecule exonuclease DNA sequencing where the continuous presence of a molecular adapter will be essential for reading individual nucleotides. Second, the ability to orient the adapter allows analytes to bind through only one of the two entrances to the betaCD cavity. We demonstrate that the covalently attached adapters can be used to alter the ion selectivity of the alphaHL pore, examine binding events at elevated temperatures, and detect analytes with prolonged dwell times.     

Controlling a single protein in a nanopore through electrostatic traps

Protein-protein pore interaction is a fundamental and ubiquitous process in biology and medical biotechnology. Here, we employed high-resolution time-resolved single-channel electrical recording along with protein engineering to examine a protein-protein pore interaction at single-molecule resolution. The pore was formed by Staphylococcus aureus alpha-hemolysin (alphaHL) protein and contained electrostatic traps formed by rings of seven aspartic acid residues placed at two different positions within the pore lumen. The protein analytes were positively charged presequences (pb2) of varying length fused to the small ribonuclease barnase (Ba). The presence of the electrostatic traps greatly enhanced the interaction of the pb2-Ba protein with the alphaHL protein pore. This study demonstrates the high sensitivity of the nanopore technique to an array of factors that govern the protein-protein pore interaction, including the length of the pb2 presequence, the position of the electrostatic traps within the pore lumen, the ionic strength of the aqueous phase, and the transmembrane potential. Alterations in the functional properties of the pb2-Ba protein and the alphaHL protein pore and systematic changes of the experimental parameters revealed the balance between forces driving the pb2-Ba protein into the pore and forces driving it out.     

Single-molecule electrophoresis of beta-hairpin peptides by electrical recordings and Langevin dynamics simulations

We used single-channel electrical recordings and Langevin molecular dynamics simulations to explore the electrophoretic translocation of various beta-hairpin peptides across the staphylococcal alpha-hemolysin (alphaHL) protein pore at single-molecule resolution. The beta-hairpin peptides, which varied in their folding properties, corresponded to the C terminal residues of the B1 domain of protein G. The translocation time was strongly dependent on the electric force and was correlated with the folding features of the beta-hairpin peptides. Highly unfolded peptides entered the pore in an extended conformation, resulting in fast single-file translocation events. In contrast, the translocation of the folded beta-hairpin peptides occurred more slowly. In this case, the beta-hairpin peptides traversed the alphaHL pore in a misfolded or fully folded conformation. This study demonstrates that the interaction between a polypeptide and a beta-barrel protein pore is dependent on the folding features of the polypeptide.     

Temperature-responsive protein pores

We describe temperature-responsive protein pores containing single elastin-like polypeptide (ELP) loops. The ELP loops were placed within the cavity of the lumen of the alpha-hemolysin (alphaHL) pore, a heptamer of known crystal structure. The cavity is roughly spherical with a molecular surface volume of about 39,500 A3. In an applied potential, the wild-type alphaHL pore remained open for long periods. In contrast, the ELP loop-containing alphaHL pores exhibited transient current blockades, the nature of which depended on the length and sequence of the inserted loop. Together with similar results obtained with poly(ethylene glycols) covalently attached within the cavity, the data suggest that the transient current blockades are caused by excursions of ELP into the transmembrane beta-barrel domain of the pore. Below its transition temperature, the ELP loop is fully expanded and blocks the pore completely, but reversibly. Above its transition temperature, the ELP is dehydrated and the structure collapses, enabling a substantial flow of ions. Potential applications of temperature-responsive protein pores in medical biotechnology are discussed.     

Single-molecule observation of the catalytic subunit of cAMP-dependent protein kinase binding to an inhibitor peptide

An engineered version of the staphylococcal alpha-hemolysin protein pore, bearing a peptide inhibitor near the entrance to the beta barrel, interacts with the catalytic (C) subunit of cAMP-dependent protein kinase. By monitoring the ionic current through the pore, binding events are detected at the single-molecule level. The kinetic and thermodynamic constants governing the binding interaction and the synergistic effect of MgATP are comparable but not identical to the values in bulk solution. Further, the values are strongly dependent on the applied membrane potential. Additional exploration of these findings may lead to a better understanding of the properties of enzymes at the lipid/water interface. Despite the complications, we suggest that the engineered pore might be used as a sensor element to screen inhibitors that act at either the substrate or ATP binding sites of the C subunit.     

Single-molecule detection of nitrogen mustards by covalent reaction within a protein nanopore

Mustards, including sulfur mustards and nitrogen mustards, form a class of cytotoxic, vesicant chemical warfare agents. Mustards have also been used to treat cancer and played a vital role in the development of chemotherapy. Additionally, because of their destructive properties, ease of synthesis, and the lack of effective antidotes, mustards are unquestionably terrorist threats. Therefore, quick and convenient detection of mustards is a critical issue. In the present study, we achieved detection of various mustards on the basis of their chemical reactivity by using engineered alpha-hemolysin (alphaHL) protein pores as sensor elements. We describe four classes of reactions for detecting mustards. These reactions occur between mustards and thiol groups contributed by cysteine side-chains within the lumen of the alphaHL pore or on an internal molecular adapter. The approach is quick and straightforward. It can confirm the existence of mustards in as little as 10 min at 50 microM or lower.     

Photoisomerization of an individual azobenzene molecule in water: an on-off switch triggered by light at a fixed wavelength

The alpha-hemolysin (alphaHL) pore was used as a nanoreactor for the direct observation of the reversible photoisomerization of individual tethered azobenzene molecules in an aqueous environment. alphaHL pores, PAZO, were used that had been derivatized within the lumen at a single cysteine residue with 4-((4-(2-chloroethanoamido)phenyl)diazenyl)benzenesulfonate. Trans-cis isomerizations were monitored at the single-molecule level by observing the modulation of the current passing through PAZO by electrical recording in planar bilayers. When PAZO was irradiated at 330 nm, continuous interconversion between the trans and cis states was observed. Either the trans or the cis state was maintained in the dark, depending upon which was present when the light source was shuttered. The cis state of PAZO was surprisingly stable in the dark, and no cis --> trans transitions were seen over a total observation period of more than 8 h. Therefore, based on our findings, it might be possible to make fast digital nanoscale switches operated by light of a fixed wavelength.     

Equivalence of two approaches for modeling ion permeation through a transmembrane channel with an internal binding site

Ion permeation through transmembrane channels has traditionally been modeled using two different approaches. In one approach, the translocation of the permeant ion through the channel pore is modeled as continuous diffusion and the rate of ion transport is obtained from solving the steady-state diffusion equation. In the other approach, the translocation of the permeant ion through the pore is modeled as hopping along a discrete set of internal binding sites and the rate of ion transport is obtained from solving a set of steady-state rate equations. In a recent work [Zhou, J. Phys. Chem. Lett. 1, 1973 (2010)], the rate constants for binding to an internal site were further calculated by modeling binding as diffusion-influenced reactions. That work provided the foundation for bridging the two approaches. Here we show that, by representing a binding site as an energy well, the two approaches indeed give the same result for the rate of ion transport.     

Encapsulating a single G-quadruplex aptamer in a protein nanocavity

The alpha-hemolysin (alphaHL) protein pore has many applications in biotechnology. This article describes a single-molecule manipulation system that utilizes the nanocavity enclosed by this pore to noncovalently encapsulate a guest molecule. The guest is the thrombin-binding aptamer (TBA) that folds into the G-quadruplex in the presence of cations. Trapping the G-quadruplex in the nanocavity resulted in characteristic changes to the pore conductance that revealed important molecular processes, including spontaneous unfolding of the quartet structure and translocation of unfolded DNA in the pore. Through detection with Tag-TBA, we localized the G-quadruplex near the entry of the beta-barrel inside the nanocavity, where the molecule vibrates and rotates to different orientations. This guest-nanocavity supramolecular system has potential for helping to understand single-molecule folding and unfolding kinetics.     

A storable encapsulated bilayer chip containing a single protein nanopore

A robust, portable chip containing a single protein nanopore would be a significant development in the practical application of stochastic sensing technology. Here, we describe a chip in which a single alpha-hemolysin (alphaHL) pore in a planar phospholipid bilayer is sandwiched between two layers of agarose gel. These encapsulated nanopore chips remain functional after storage for weeks. The detection of the second messenger inositol 1,4,5-trisphosphate (IP3) was demonstrated with a chip containing a genetically engineered alphaHL pore as the sensor element.     

Effect of charge distribution on the translocation of an inhomogeneously charged polymer through a nanopore

We investigate the voltage-driven translocation of an inhomogeneously charged polymer through a nanopore by utilizing discrete and continuous stochastic models. As a simplified illustration of the effect of charge distribution on translocation, we consider the translocation of a polymer with a single charged site in the presence and absence of interactions between the charge and the pore. We find that the position of the charge that minimizes the translocation time in the absence of pore-polymer interactions is determined by the entropic cost of translocation, with the optimum charge position being at the midpoint of the chain for a rodlike polymer and close to the leading chain end for an ideal chain. The presence of attractive and repulsive pore-charge interactions yields a shift in the optimum charge position toward the trailing end and the leading end of the chain, respectively. Moreover, our results show that strong attractive or repulsive interactions between the charge and the pore lengthen the translocation time relative to translocation through an inert pore. We generalize our results to accommodate the presence of multiple charged sites on the polymer. Our results provide insight into the effect of charge inhomogeneity on protein translocation through biological membranes.     

Polymer capture by electro-osmotic flow of oppositely charged nanopores

The authors have addressed theoretically the hydrodynamic effect on the translocation of DNA through nanopores. They consider the cases of nanopore surface charge being opposite to the charge of the translocating polymer. The authors show that, because of the high electric field across the nanopore in DNA translocation experiments, electro-osmotic flow is able to create an absorbing region comparable to the size of the polymer around the nanopore. Within this capturing region, the velocity gradient of the fluid flow is high enough for the polymer to undergo coil-stretch transition. The stretched conformation reduces the entropic barrier of translocation. The diffusion limited translocation rate is found to be proportional to the applied voltage. In the authors' theory, many experimental variables (electric field, surface potential, pore radius, dielectric constant, temperature, and salt concentration) appear through a single universal parameter. They have made quantitative predictions on the size of the adsorption region near the pore for the polymer and on the rate of translocation.     

AC conductance of transmembrane protein channels. The number of ionized residue mobile counterions at infinite dilution

Simultaneous measurements of the AC and DC conductances of alpha-hemolysin (alphaHL) ion channels and outer membrane protein F (OmpF) porins in dilute ionic solutions is described. AC conductance measurements were performed by applying a 10 mV rms AC voltage across a suspended planar bilayer of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine in the absence and presence of the protein and detecting the AC current response using phase-sensitive lock-in techniques. The conductances of individual alphaHL channels and OmpF porins were measured in symmetric KCl solutions containing between 5 and 1000 mM KCl. The AC and DC conductances of each protein were in agreement for all solution conditions, demonstrating the reliability of the AC method in single-channel recordings. Linear plots of conductance versus bulk KCl concentration for both proteins extrapolate to significant nonzero conductances (0.150 +/- 0.050 nS and 0.028 +/- 0.008 nS for OmpF and alphaHL, respectively) at infinite KCl dilution. The infinite dilution conductances are ascribed to mobile counterions of the ionizable residues within the protein lumens. A method of analyzing the plots of conductance vs KCl concentration is introduced that allows the determination of the concentration of mobile counterions associated with ionizable groups without knowledge of either the protein geometry or the ion mobilities. At neutral pH, an equivalent of 3 mobile counterions (K+ or Cl-) is estimated to contribute to the conductivity of the alphaHL channel.     

Subdiffusion as a model of transport through the nuclear pore complex

Cargo transport through the nuclear pore complex continues to be a subject of considerable interest to experimentalists and theorists alike. Several recent studies have revealed details of the process that have still to be fully understood, among them the apparent nonlinearity between cargo size and the pore crossing time, the skewed, asymmetric nature of the distribution of such crossing times, and the non-exponentiality in the decay profile of the dynamic autocorrelation function of cargo positions. In this paper, we show that a model of pore transport based on subdiffusive particle motion is in qualitative agreement with many of these observations. The model corresponds to a process of stochastic binding and release of the particle as it moves through the channel. It suggests that the phenylalanine-glycine repeat units that form an entangled polymer mesh across the channel may be involved in translocation, since these units have the potential to intermittently bind to hydrophobic receptor sites on the transporter protein.     

Stochastic sensing of nanomolar inositol 1,4,5-trisphosphate with an engineered pore

The introduction of a ring of arginine residues near the constriction in the transmembrane beta barrel of the staphylococcal alpha-hemolysin heptamer yielded a pore that could be almost completely blocked by phosphate anions at pH 7.5. Block did not occur with other oxyanions, including nitrate, sulfate, perchlorate, and citrate. Based on this finding, additional pores were engineered with high affinities for important cell signaling molecules, such as the Ca(2+)-mobilizing second messenger inositol 1,4,5-trisphosphate (IP(3)), that contain phosphate groups. One of these engineered pores, P(RR-2), provides a ring of fourteen arginines that project into the lumen of the transmembrane barrel. Remarkably, P(RR-2) bound IP(3) with low nanomolar affinity while failing to bind another second messenger, adenosine 3', 5'-cyclic monophosphate (cAMP). The engineered alpha-hemolysin pores may be useful as components of stochastic sensors for cell signaling molecules.     

Free energy calculations on the two drug binding sites in the M2 proton channel

Two alternative binding sites of adamantane-type drugs in the influenza A M2 channel have been suggested, one with the drug binding inside the channel pore and the other with four drug molecule S-binding to the C-terminal surface of the transmembrane domain. Recent computational and experimental studies have suggested that the pore binding site is more energetically favorable but the external surface binding site may also exist. Nonetheless, which drug binding site leads to channel inhibition in vivo and how drug-resistant mutations affect these sites are not completely understood. We applied molecular dynamics simulations and potential of mean force calculations to examine the structures and the free energies associated with these putative drug binding sites in an M2-lipid bilayer system. We found that, at biological pH (~7.4), the pore binding site is more thermodynamically favorable than the surface binding site by ~7 kcal/mol and, hence, would lead to more stable drug binding and channel inhibition. This result is in excellent agreement with several recent studies. More importantly, a novel finding of ours is that binding to the channel pore requires overcoming a much higher energy barrier of ~10 kcal/mol than binding to the C-terminal channel surface, indicating that the latter site is more kinetically favorable. Our study is the first computational work that provides both kinetic and thermodynamic energy information on these drug binding sites. Our results provide a theoretical framework to interpret and reconcile existing and often conflicting results regarding these two binding sites, thus helping to expand our understanding of M2-drug binding, and may help guide the design and screening of novel drugs to combat the virus.     

Local Unfolding of Fatty Acid Binding Protein to Allow Ligand Entry for Binding

Fatty acid binding proteins are responsible for the transportation of fatty acids in biology. Despite intensive studies, the molecular mechanism of fatty acid entry to and exit from the protein cavity is still unclear. Here a cap‐closed variant of human intestinal fatty acid binding protein was generated by mutagenesis, in which the helical cap is locked to the β‐barrel by a disulfide linkage. Structure determination shows that this variant adopts a closed conformation, but still uptakes fatty acids. Stopped‐flow experiments indicate that a rate‐limiting step exists before the ligand association and this step corresponds to the conversion of the closed form to the open one. NMR relaxation dispersion and H‐D exchange data demonstrate the presence of two excited states: one is native‐like, but the other adopts a locally unfolded structure. Local unfolding of helix 2 generates an opening for ligands to enter the protein cavity, and thus controls the ligand association rate.     

Polymer translocation through a nanopore. II. Excluded volume effect

Following our previous study of a Gaussian chain translocation, we have investigated the transport of a self-avoiding chain from one sphere to another sphere through a narrow pore, using the self-consistent field theory formalism. The free energy landscape for polymer translocation is significantly modified by excluded volume interactions among monomers. The free energy barrier for the placement of one of the chain ends at the pore depends on the chain length N nonmonotonically, in contrast to the N-independence for Gaussian chains. This results in a nonmonotonic dependence of the average arrival time [tau0] on N for self-avoiding chains. When the polymer chain is partitioned between the donor and recipient spheres, a local free energy minimum develops, depending on the strength w of the excluded volume interaction and the relative sizes of the donor and recipient spheres. If the sizes of spheres are comparable, the average translocation time tau (the average time taken by the polymer, after the arrival at the pore, to convert from the donor to the recipient) increases with an increase in w for a fixed N value. On the other hand, for the highly asymmetric sizes of the donor and recipient spheres, tau decreases with an increase in w. As in the case of Gaussian chains, tau depends nonmonotonically on the pore length.     

Cooperative motions of protein and hydration water molecules: molecular dynamics study of scytalone dehydratase

Two molecular dynamics (MD) simulations totaling 25 ns of simulation time of monomeric scytalone dehydratase (SD) were performed. The enzyme has a ligand-binding pocket containing a cone-shaped alpha+beta barrel, and the C-terminal region covers the binding pocket. Our simulations clarified the difference in protein dynamics and conformation between the liganded protein and the unliganded protein. The liganded protein held the ligand molecule tightly and the initial structure was maintained during the simulation. The unliganded protein, on the other hand, fluctuated dynamically and its structure changed largely from the initial structure. In the equilibrium state, the binding pocket was fully solvated by opening of the C-terminal region, and the protein dynamics was connected with hydration water molecules entry into and release from the binding pocket. In addition, the cooperative motions of the unliganded protein and the hydration water molecules produced the path through the protein interior for ligand binding.     

Fluorescence recovery after photobleaching reveals the biochemistry of nucleocytoplasmic exchange

Fluorescence recovery after photobleaching (FRAP) can help unveil subtle dynamical and biochemical properties of intracellular components. A peculiar aspect of this method is that it is based on the change of optical properties only, whereas dynamics and biochemistry of the molecules of interest are not perturbed. This makes FRAP particularly suitable for the study of protein translocation, e.g., between nucleus and cytoplasm. Here we present a comprehensive theoretical treatment of FRAP applied to protein nucleocytoplasmic translocation by passive diffusion and/or energy-driven processes across the nuclear envelope. Our mathematical model is validated by experimental FRAP studies with functionalized fluorescent protein chimeras. Using this approach we demonstrate that molecular crowding at the nuclear pore does not hamper passive diffusion and calculate the dimension of the nuclear pore size (5.33 nm). Additionally, our FRAP analysis reveals the biochemical parameters (maximum translocation rate and dissociation constant of the transport complex in cytoplasm) associated with the active import of a prototypical nuclear localization sequence (NLS of SV40) and related mutants. We demonstrate that transportin binding and active import into the nucleus are independent processes that can be separately modulated. The present results are discussed in light of their potential to help in engineering sequences for intracellular targeted delivery of sensors and/or therapeutic compounds. Finally, the limits of validity of our mathematical model are addressed.