Structure and energetics of channel-forming protein-polysaccharide complexes inferred via computational statistical thermodynamics

The ion channel protein alpha-hemolysin (alphaHL) forms supramolecular complexes with the polysaccharide beta-cyclodextrin (betaCD). This system has potential uses in nanoscale device engineering. It has been found recently that betaCD formed longer- or shorter-lived complexes with some engineered alphaHL mutants then with a wild type protein (Gu et al. J. Gen. Physiol. 2001, 118, 481-493). However, how changes in the protein sequence affect complex lifetime was not completely understood in part due to the lack of knowledge of structures of these metastable complexes. In this paper, we present an extensive molecular modeling study of the betaCD-alphaHL and selected mutant complexes to gain insights into the betaCD-alphaHL interaction mechanisms and to predict possible structures and energetics of the complexes. Thermodynamic integration (TI) and umbrella sampling (US) techniques (with the weighted histogram analysis method (WHAM)) were used to calculate the relative binding affinities of the complexes formed with the wild type alphaHL and the M113N, M113E, M113A, and M113V mutants. Our results are in excellent agreement with experiment. While betaCD-M113N and betaCD-M113A complexes were stable in the configuration of the wild type complex, the equilibrium configuration of the betaCD-M113V and betaCD-M113E complexes was significantly different. In these cases, TI alone was insufficient to accurately calculate the corresponding free energy differences. By utilizing a TI/US combination in a novel manner, we were able to accurately calculate free energy changes in these flexible systems. The betaCD-M113A and betaCD-M113E complexes, which exhibited shorter lifetimes than other complexes in an experiment, in simulations exhibited greater flexibility and higher water solvation of the betaCD adapter. MD simulations of the betaCD-M113N complex with betaCD in a downward orientation were also performed.     

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.     

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.     

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.     

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 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.     

Programmable Supra‐Assembly of a DNA Surface Adapter for Tunable Chiral Directional Self‐Assembly of Gold Nanorods

An important challenge in molecular assembly and hierarchical molecular engineering is to control and program the directional self‐assembly into chiral structures. Here, we present a versatile DNA surface adapter that can programmably self‐assemble into various chiral supramolecular architectures, thereby regulating the chiral directional “bonding” of gold nanorods decorated by the surface adapter. Distinct optical chirality relevant to the ensemble conformation is demonstrated from the assembled novel stair‐like and coil‐like gold nanorod chiral metastructures, which is strongly affected by the spatial arrangement of neighboring nanorod pair. Our strategy provides new avenues for fabrication of tunable optical metamaterials by manipulating the directional self‐assembly of nanoparticles using programmable surface adapters.     

Catalyzing the translocation of polypeptides through attractive interactions

Facilitated translocation of polypeptides through a protein pore is a ubiquitous and fundamental process in biology. Several translocation systems possess various well-defined binding sites within the pore lumen, but a clear mechanistic understanding of how the interaction of the polypeptides with the binding site alters the underlying kinetics is still missing. Here, we employed rational protein design and single-channel electrical recordings to obtain detailed kinetic signatures of polypeptide translocation through the staphylococcal alpha-hemolysin (alphaHL) transmembrane pore, a robust, tractable, and versatile beta-barrel protein. Acidic binding sites composed of rings of negatively charged aspartic acid residues, engineered at strategic positions within the beta barrel, produced dramatic changes in the functional properties of the alphaHL protein, facilitating the transport of cationic polypeptides from one side of the membrane to the other. When two electrostatic binding sites were introduced, at the entry and exit of the beta barrel, both the rate constants of association and dissociation increased substantially, diminishing the free energy barrier for translocation. By contrast, more hydrophobic polypeptides exhibited a considerable decrease in the rate constant of association to the pore lumen, having to overcome a greater energetic barrier because of the hydrophilic nature of the pore interior.     

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.     

How does beta-cyclodextrin affect oxygen solubility in aqueous solutions of sodium perfluoroheptanoate?

The solubility of oxygen in aqueous solutions of sodium perfluoroheptanoate (NaPFHept) at different concentrations was measured at 310.15 K with an apparatus based on the saturation method. The effect of adding beta-cyclodextrin (betaCD) on the solubility of oxygen was also studied. Conductimetry measurements showed that the presence of betaCD in aqueous solutions of NaPFHept increases its critical micellar concentration (CMC). In the presence of betaCD (15 mM), the characteristic minimum of oxygen solubility observed at the CMC is shifted from 83 to 114 mM, and the curvature at the minimum is reduced to 64% of the value in the absence of betaCD. Chemical shift changes for the H5 protons of betaCD, recorded as functions of the initial concentration of NaPFHept, point to the formation of a relatively strong 1:1 inclusion in betaCD of the perfluoroheptanoate anion. Hence, it is suggest that the effect of adding betaCD on the solubility of oxygen cannot be accounted for only by the perfluoroheptanoate anion inclusion in betaCD, but has to be ascribed to the direct influence of this inclusion complex on disrupting the aggregation process reducing the increase of oxygen solubility after the CMC value.     

Toward single molecule DNA sequencing: direct identification of ribonucleoside and deoxyribonucleoside 5'-monophosphates by using an engineered protein nanopore equipped with a molecular adapter

Individual nucleic acid molecules might be sequenced by the identification of nucleoside 5'-monophosphates as they are released by processive exonucleases. Here, we show that single molecule detection with a modified protein nanopore can be used to identify ribonucleoside and 2'-deoxyribonucleoside 5'-monophosphates, thereby taking a step along this path. Distinct levels of current block are observed for each of the four members of a set of nucleoside 5'-monophosphates when the molecules bind within a mutant alpha-hemolysin pore, (M113R)(7), equipped with the molecular adapter heptakis-(6-deoxy-6-amino)-beta-cyclodextrin. While our results compare favorably with alternative approaches, further work will be required to improve the accuracy of identification of the nucleic acid bases, to feed each released nucleotide into the pore, and to ensure that every nucleotide is captured by the adapter.     

Simulations of stochastic sensing of proteins

We have performed Langevin dynamics and Poisson-Nernst-Planck calculations to simulate detection of proteins by genetically engineered alpha-hemolysin channels. In the recent stochastic sensing experiments, one end of a flexible polymer chain is permanently anchored inside the protein channel at a specified location, and the other end undergoes complexation with an analyte. Our simulations, using coarse-grained modeling, reproduce all essential qualitative results of the electrophysiology measurements of stochastic sensing. In addition, the underlying macromolecular mechanisms behind stochastic sensing are revealed in vivid details. The entropic fluctuations of the conformations of the tethered polymer chain dictate crucially the unique signatures of the ionic current trace of the channel and provide design rules for successful stochastic sensing. The origin of strong fluctuations in the ionic current of the channel is found to arise from the obstruction of the entrance at the beta-barrel of the channel by the fluctuating segments of the tether. Silencing of the pore is due to the suppression of conformational fluctuations of the chain, and the permanent blockade of ionic current is due to the threading of the tether through the channel. The onset of silencing and permanent blockade of the channel current cannot necessarily be attributed to the capture of analytes. In order for detection events to be timed accurately, the length and anchoring location of the tether must be tuned appropriately.     

Stochastic detection of enantiomers

The rapid quantification of the enantiomers of small chiral molecules is very important, notably in pharmacology. Here, we show that the enantiomers of drug molecules can be distinguished by stochastic sensing, a single-molecule detection technique. The sensing element is an engineered alpha-hemolysin protein pore, fitted with a beta-cyclodextrin adapter. By using the approach, the enantiomeric composition of samples of ibuprofen and thalidomide can be determined in less than 1 s.     

Protein detection by nanopores equipped with aptamers

Protein nanopores have been used as stochastic sensors for the detection of analytes that range from small molecules to proteins. In this approach, individual analyte molecules modulate the ionic current flowing through a single nanopore. Here, a new type of stochastic sensor based on an αHL pore modified with an aptamer is described. The aptamer is bound to the pore by hybridization to an oligonucleotide that is attached covalently through a disulfide bond to a single cysteine residue near a mouth of the pore. We show that the binding of thrombin to a 15-mer DNA aptamer, which forms a cation-stabilized quadruplex, alters the ionic current through the pore. The approach allows the quantification of nanomolar concentrations of thrombin, and provides association and dissociation rate constants and equilibrium dissociation constants for thrombin·aptamer interactions. Aptamer-based nanopores have the potential to be integrated into arrays for the parallel detection of multiple analytes.     

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.     

Dynamic Covalent Nanoparticle Building Blocks

Rational and generalisable methods for engineering surface functionality will be crucial to realising the technological potential of nanomaterials. Nanoparticle‐bound dynamic covalent exchange combines the error‐correcting and environment‐responsive features of equilibrium processes with the stability, structural precision, and vast diversity of covalent chemistry, defining a new and powerful approach for manipulating structure, function and properties at nanomaterial surfaces. Dynamic covalent nanoparticle (DCNP) building blocks thus present a whole host of possibilities for constructing adaptive systems, devices and materials that incorporate both nanoscale and molecular functional components. At the same time, DCNPs have the potential to reveal fundamental insights regarding dynamic and complex chemical systems confined to nanoscale interfaces.     

A biocompatible surfactant with folded hydrophilic head group: enhancing the stability of self-inclusion complexes of ferrocenyl in a beta-cyclodextrin unit by bond rigidity

This work reports a new biocompatible surfactant structure, of which the hydrophilic head group is composed of a folded, stable self-inclusion complex of a ferrocenyl substituted beta-cyclodextrin (betaCD). While multiple intra- or intermolecular complexes can exist for this amphiphile, the molecule folds into a unique intramolecular complex with well-defined conformation, in which part of the aliphatic chain and the ferrocene group are both included in the annular cavity of betaCD. Study of different isosteric covalent linkages indicates that this folded structure is stable against displacement by the presence of other small guest molecules. Furthermore, in contrast to ferrocene-CD conjugates that are without the aliphatic chain, the presence of small guest molecules in solution does not influence at all the induced circular dichroism signal of this amphiphile, indicating a sterically congested, but stable, folded conformation of the inclusion complex. This new amphiphile is surface active and, more importantly, does not denature the membrane protein bacteriorhodopsin. Finally, because this surfactant forms self-assembled aggregates, this work introduces a folded structure into soft matters formed by amphiphiles in water.     

Controlling the supramolecular assembly of redox-active dendrimers at molecular printboards by scanning electrochemical microscopy

Redox-active ferrocenyl (Fc)-functionalized poly(propylenimine) (PPI) dendrimers solubilized in aqueous media by complexation of the Fc end groups with beta-cyclodextrin (betaCD) were immobilized at monolayers of betaCD on glass ("molecular printboards") via multiple host-guest interactions. The directed immobilization of the third-generation dendrimer-betaCD assembly G3-PPI-(Fc)16-(betaCD)16 at the printboard was achieved by supramolecular microcontact printing. The redox activity of the patterned dendrimers was mapped by scanning electrochemical microscopy (SECM) in the positive feedback mode using [IrCl(6)](3-) as a mediator. Local oxidation of the Fc-dendrimers by the microelectrode-generated [IrCl(6)](2-) resulted in an effective removal of the Fc-dendrimers from the host surface since the oxidation of Fc to the oxidized form (Fc+) leads to a concomitant loss of affinity for betaCD. Thus, SECM provided a way not only to image the surface, but also to control the binding of the Fc-terminated dendrimers at the molecular printboard. Additionally, the electrochemical desorption process could be monitored in time as the dendrimer patterns were gradually erased upon multiple scans.     

衍生多孔碳材料在固相微萃取中的应用研究进展

固相微萃取是一种集采样、萃取、富集和进样于一体的样品前处理技术,其萃取效果与涂层材料密切相关。多孔碳材料具有比表面积大、多孔结构可控、活性位点多和化学稳定性好等优点,广泛应用于电池、超级电容器、催化、吸附和分离等领域,也是一种热门的用作固相微萃取探针的涂层材料。衍生多孔碳材料因种类丰富、可设计性强被广泛研究,研究主要集中在对衍生多孔碳材料的结构优化方面。但是衍生多孔碳材料在固相微萃取中的应用还存在如下问题:(1)共价有机框架衍生多孔碳材料的制备已取得较大进展,但将其应用于固相微萃取领域的研究仍较少;(2)有待进一步明确制备出的衍生多孔碳材料用作固相微萃取涂层表现出优异提取能力的机理;(3)有待进一步深入研究将衍生多孔碳材料用作固相微萃取涂层以实现对不同物理化学性质污染物的广谱高灵敏度分析。文章综述了近3年衍生多孔碳材料在固相微萃取中的应用研究,并展望了未来衍生多孔碳材料在固相微萃取中的研究前景。引用文献共56篇,主要来源于Elsevier。     

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.