The cavity within the transmembrane staphylococcal α-hemolysin (αHL) pore is roughly a sphere of diameter ~45 Å (volume ~32,600 Å3). The alpha-hemolysin gene was modified to introduce exogenous polypeptide sequences between positions 105 and 106 of αHL. These modified αHLs were assembled either by themselves or with wild-type (W) subunits to form stable homoheptamers and heteroheptamers, respectively. First, the ability to accommodate Gly/Ser-rich polypeptide sequences in the central cavity was tested. Concatemerized Gly/Ser-containing sequences ("loops", L; L(10n + 5), n = 0 to 21) were inserted by genetic approaches. Detailed analysis of bilayer recordings and electrophoretic migration patterns of assembled pores indicate that the upper capacity of the cavity is ~175 amino acids. Then two different polypeptides were placed in the cavity to introduce novel functional properties to the αHL pore. By introducing tandem repeats of elastin-like polypeptide sequences (VPGGG), αHL pores (E101W6) that featured a temperature-responsive gating mechanism were obtained. The temperature-dependent properties of E101W6 pores were monitored by single-channel current recording in planar lipid bilayers. The amplitude and the frequency of the transient blockades increased as the temperature increased, while their duration decreased. The hydrophobic collapse of the inserted ELP loop is proposed for the source of the observed sigmoidal two-state transition for normalized closed states of E101W6 pores. Lastly, an αHL pore was designed to detect proteins from the cis side of the membrane. The heat-stable protein kinase inhibitor (PKI) sequence was inserted into the mid-position of the Gly/Ser loop, which was generated by previous project (L105 construct). The heteromeric pore with the PKI-containing loop (P1151W6) was able to detect cAMP-dependent protein kinase catalytic subunit (PKA) at single molecular level. These engineered αHL pores provide numerous possibilities as tools for drug delivery, cryopreservation, or molecular sensing.

The cavity within the transmembrane staphylococcal α-hemolysin (αHL) pore is roughly a sphere of diameter ~45 Ã (volume ~32,600 Ã 3). The alpha-hemolysin gene was modified to introduce exogenous polypeptide sequences between positions 105 and 106 of αHL. These modified αHLs were assembled either by themselves or with wild-type (W) subunits to form stable homoheptamers and heteroheptamers, respectively. First, the ability to accommodate Gly/Ser-rich polypeptide sequences in the central cavity was tested. Concatemerized Gly/Ser-containing sequences ("loops", L; L(10n + 5), n = 0 to 21) were inserted by genetic approaches. Detailed analysis of bilayer recordings and electrophoretic migration patterns of assembled pores indicate that the upper capacity of the cavity is ~175 amino acids. Then two different polypeptides were placed in the cavity to introduce novel functional properties to the αHL pore. By introducing tandem repeats of elastin-like polypeptide sequences (VPGGG), αHL pores (E101W6) that featured a temperature-responsive gating mechanism were obtained. The temperature-dependent properties of E101W6 pores were monitored by single-channel current recording in planar lipid bilayers. The amplitude and the frequency of the transient blockades increased as the temperature increased, while their duration decreased. The hydrophobic collapse of the inserted ELP loop is proposed for the source of the observed sigmoidal two-state transition for normalized closed states of E101W6 pores. Lastly, an αHL pore was designed to detect proteins from the cis side of the membrane. The heat-stable protein kinase inhibitor (PKI) sequence was inserted into the mid-position of the Gly/Ser loop, which was generated by previous project (L105 construct). The heteromeric pore with the PKI-containing loop (P1151W6) was able to detect cAMP-dependent protein kinase catalytic subunit (PKA) at single molecular level. These engineered αHL pores provide numerous possibilities as tools for drug delivery, cryopreservation, or molecular sensing.

Pathogenic bacteria secrete various virulence factors as their biochemical weapons to gain access to and destroy the target cells. They can directly interact with the outer lipid bilayer membrane of eukaryotic cells, inducing the premature cell death by either apoptosis or necrosis. Such virulence factors account for much of the toxic actions associated with bacterial infection; therefore the detection of such proteins could provide a methodology for sensing/detection of pathogenic bacteria in, for example, food or human tissue. Detection and identification of pathogenic bacteria by conventional methods such as plating and counting in laboratory is expensive and time consuming. With growing concerns over emergence and re-emergence of pathogenic bacteria with high resistant to current antibiotics, there is a potential need for effective detection of pathogenic toxins invitro. On the other hand, artificially prepared lipid bilayer membrane on planar metallic surfaces provides the cell membrane mimics which are extremely useful in exploring the cellular functions and processes at the molecular level. Therefore in this work, an application of planar tethered bilayer lipid membrane (pTBLM) as a biomimetic sensing platform for the detection of clinically important pathogens, Staphylococcus aureus and Pseudomonas aeruginosa via their secreted virulence factors was presented. Planar TBLM was modified by incorporation of cholesterol and detection of bacterial toxins at human body temperature was examined by impedance and surface plasmon resonance methods. The results of pathogenic bacterial toxin detection were compared with those of Escherichia coli (DH5α), the human gut normal flora with non-pathogenic strain, as a control. Additionally pTBLM was transferred onto single nanoporous Si3N4 membrane to enhance the toxin sensitivity and extend the lifetime for the possible realization of future membrane chips for ion channel characterizations and drug screenings. Then the single ion channel measurement was demonstrated with nanopore-suspended TBLM (Nano-psTBLM) using α-toxin of S. aureus. The results presented in this work therefore, may pave the more effective and efficient ways for future pathogenic bacterial detection in which the sensing mechanism was solely based on the nature of interactions as well as modes of action between bacterial toxins and artificial lipid bilayer membranes.

Single-molecule detection has provided insights into how molecules behave. Without the averaging effect of ensemble measurements, the stochastic behaviour of single molecules can be observed and intermediate steps in multistep transformations can be clearly detected. The single-molecule reactants range from small molecules (e.g. propene) to proteins of several tens of kDa (e.g. myosin). One single-molecule detection technique is single-channel electrical recording. This approach is based on the measurement of the transmembrane ionic current flowing through a nanoscale transmembrane pore under an applied potential. In this thesis, the protein α-hemolysin was employed as a nanoreactor. α-Hemolysin is a toxin secreted by Staphylococcus aureus. Its transmembrane pore (~100 Å in length and ≥14 Å in diameter) allows ions, water and small molecules to pass through its lumen. Under an applied potential, chemical changes in reactants attached to the internal wall of the pore modulate the flow of ions, leading to changes in the transmembrane ionic current. Analysis of this current provides information about the reaction kinetics and mechanisms. Chapter 1 – Single-Molecule Chemistry and α-Hemolysin is an introductory chapter that is divided into two parts. Section 1.1 provides an overview of the different techniques for the detection of chemical reactions at the single-molecule level. Section 1.2 gives a brief review of the protein pore α-hemolysin, including its structure, properties and various applications. Chapter 2 – S-Nitrosothiol Chemistry applies cysteine-containing α-hemolysins to study the biologically relevant chemistry of S-nitrosothiols (RSNO). RSNO are important molecules involved in cell signalling, which control physiological processes such as vasodilation and bronchodilation. Three reactions, namely transnitrosation (the transfer of the ‘NO’ group from RSNO to a thiol), S-thiolation (the formation of a disulfide from RSNO and thiol) and S-sulfonation (the generation of an S-sulfonate (RSSO₃⁻) from RSNO and sulfite ion), were investigated at the single-molecule level. The pH-dependency of the two competing reactions (transnitrosation and S-thiolation), the lifetime of the proposed transnitrosation intermediate, and nature of the chemical reaction between RSNO and sulfite (a bronchoconstrictor) were determined. Chapter 3 – Silver(I)-thiolate and cadmium(II)-thiolate complexes describes the kinetics of the formation and breakdown of these two metal-thiolate complexes. Ag⁺ and Cd²⁺ are commonly used in probing the membrane topology and gating properties of ion channels using the scanning cysteine accessibility method (SCAM). The binding of two Ag⁺ ions per thiol group and the stepwise build-up and dissociation of Cd²⁺-glutathione complexes were unambiguously characterized. Chapter 4 – Copper(II)-Catalyzed Diels-Alder Reactions reports the attempt to carry out copper(II)-catalyzed Diels-Alder reactions inside an engineered α-hemolysin. An iminodiacetate ligand was covalently attached within the lumen of the α-hemolysin pore. This ligand chelates Cu²⁺ ion, which can bind bidentate dienophiles and activate them towards Diels-Alder reaction with dienes. However, due to the ‘slow’ reaction rate of the Diels-Alder reaction (rate constant ~10⁻¹ M⁻¹s⁻) relative to the time-scale of the single-molecule experiment, we failed to observed chemical conversion at the single-molecule level. Nevertheless, the engineered metal-binding α-hemolysin may be useful for sensing molecules bearing metal-coordinating groups.

Metallic quantum clusters are stable structures that can exhibit many useful magnetic, chemical, and optical properties. Developing clusters for specific applications requires accurate methods for characterizing their physical and chemical properties. Most cluster characterization methods are ensemble-based measurements that can only measure the average values of the cluster properties. Single cluster measurements improve upon this by yielding information about the distribution of cluster parameters. This investigation describes the initial results on a new approach to detecting and characterizing individual gold nanoclusters (Au25(SG)18) in an aqueous solution with nanopore-based resistive pulse sensing. We also present a new application where the clusters are shown to increase the mean residence time of polyethylene glycol (PEG) molecules within an alpha hemolysin (αHL) nanopore. The effect appears over a range of PEG sizes and ionic strengths. This increases the resolution of the peaks in the single molecule mass spectrometry (SMMS) current blockade distribution and suggests a means for reducing the ionic strength of the nanopore solute in the SMMS protocol.

In nanopore analysis, peptides and proteins can be detected by the change in current when single molecules interact with an α-hemolysin pore embedded in a lipid membrane. Studies into the effects of fluorenylmethoxycarbonyl (Fmoc), acetylation or proline modification to negatively charged α-helical peptides showed that Fmoc peptides give more translocations than acetylated peptides. The addition of a proline in the middle of an acetylated peptide further reduces the number of translocations compared to Fmoc. The effect of peptide conformation on translocation or intercalation was studied with small α-helical and β-sheet hairpins. The capped β-hairpin increased translocations compared to the uncapped. The Fmoc-α-helical hairpin, containing a disulfide link, displayed both bumping and translocations whereas in the unlinked peptide the proportion of translocations was greater. Prion diseases arise from the misfolding and aggregation of the normal cellular prion protein. Nanopore analysis of prion peptides with α-helical and β-strand sequences show changes to the event parameters that help distinguish them. The interaction of bovine prion protein (bPrP), with α-hemolysin showed both bumping (type-I) and intercalation/translocation (type-II) events. There are several lines of evidence that indicate these type-II events with a blockade current of -65 pA for bPrP, represent translocations. Nanopore analysis showed that about 37% events were translocations. The interaction of metal ions with bPrP showed that Cu(II) or Zn(II) reduced translocations. Surprisingly, Mn(II) caused an increase in translocation events to about 64%. Complex formation between antibodies and prion peptides and proteins can be detected by nanopore analysis. The PrP/antibody complex is too large to translocate whereas the event parameters for unbound molecules are unchanged. In principle, a nanopore can detect a single molecule; thus, this work represents the first step towards the development of a prion detector. The nanopore will provide the sensitivity and the antibodies will provide the specificity to distinguish between PrPC and PrPSc. Also, the prion N- and C-terminal signal peptides interact with bPrP changing the event parameters, relating to a new mechanism. Finally, the folding intermediates of bPrP at 0.86 M Gdn-HCl suggests that the protein unfolds and then refolds into a different conformation with event parameters similar to those of bPrP.

In nanopore analysis, peptides and proteins can be detected by the change in current when single molecules interact with an α-hemolysin pore embedded in a lipid membrane. Studies into the effects of fluorenylmethoxycarbonyl (Fmoc), acetylation or proline modification to negatively charged α-helical peptides showed that Fmoc peptides give more translocations than acetylated peptides. The addition of a proline in the middle of an acetylated peptide further reduces the number of translocations compared to Fmoc. The effect of peptide conformation on translocation or intercalation was studied with small α-helical and β-sheet hairpins. The capped β-hairpin increased translocations compared to the uncapped. The Fmoc-α-helical hairpin, containing a disulfide link, displayed both bumping and translocations whereas in the unlinked peptide the proportion of translocations was greater. Prion diseases arise from the misfolding and aggregation of the normal cellular prion protein. Nanopore analysis of prion peptides with α-helical and β-strand sequences show changes to the event parameters that help distinguish them. The interaction of bovine prion protein (bPrP), with α-hemolysin showed both bumping (type-I) and intercalation/translocation (type-II) events. There are several lines of evidence that indicate these type-II events with a blockade current of -65 pA for bPrP, represent translocations. Nanopore analysis showed that about 37% events were translocations. The interaction of metal ions with bPrP showed that Cu(II) or Zn(II) reduced translocations. Surprisingly, Mn(II) caused an increase in translocation events to about 64%. Complex formation between antibodies and prion peptides and proteins can be detected by nanopore analysis. The PrP/antibody complex is too large to translocate whereas the event parameters for unbound molecules are unchanged. In principle, a nanopore can detect a single molecule; thus, this work represents the first step towards the development of a prion detector. The nanopore will provide the sensitivity and the antibodies will provide the specificity to distinguish between PrPC and PrPSc. Also, the prion N- and C-terminal signal peptides interact with bPrP changing the event parameters, relating to a new mechanism. Finally, the folding intermediates of bPrP at 0.86 M Gdn-HCl suggests that the protein unfolds and then refolds into a different conformation with event parameters similar to those of bPrP.

No description available.

The covalent chemistry of individual reactants bound within a protein nanopore can be monitored by observing the ionic current flow through the pore, which acts as a nanoreactor responding to bond-making and bond-breaking events. However, chemistry investigated in this way has been largely confined to the reactions of thiolates, presented by the side chains of cysteine residues. The introduction of unnatural amino acids would provide a large variety of reactive side chains with which additional single-molecule chemistry could be investigated. An efficient method to incorporate unnatural amino acid is semisynthesis, which allows site-specific modification with a chemically-defined functional group. However, relatively little work has been done on engineered membrane proteins. This deficiency stems from attributes inherent to proteins that interact with lipid bilayer, notably the poor solubility in aqueous buffer. In the present work, four different derivatives α-hemolysin (αHL) monomer were obtained either by two- or three-way native chemical ligation. The semisynthetic αHL monomers were successfully refolded to heptameric pores and used as nanoreactors to study single-molecule chemistry. The semisynthetic pores show similar biophysical properties to native αHL pores obtained from an in vitro transcription and translation technique. Interestingly, when αHL pores with one semisynthetic subunit containing a terminal alkyne group were used to study Cu(I)-catalyzed azide-alkyne cycloaddition, a long-lived intermediate in the reaction was directly observed.

Staphylococcal leukocidin pores are formed by the obligatory interaction of two distinct polypeptides, one of class F and one of class S, making them unique in the family of β-barrel pore-forming toxins (β-PFTs). By contrast, other β-PFTs form homooligomeric pores. For example, the staphylococcal α- hemolysin is a homoheptamer. Limited and controversial data exist on the assembly and molecular architecture of the leukocidin pore. In this work, biochemical and biophysical methods were used to characterize the leukocidin pore produced by the LukF (HlgB) and LukS (HlgC) components encoded by Staphylococcus aureus. I demonstrate that LukF and LukS assemble to form an SDS-stable pore on rabbit erythrocyte membranes. In addition, the pore-forming properties of recombinant leukocidin were investigated with planar lipid bilayers. Although leukocidins and staphylococcal α-hemolysin share partial sequence identity and related folds, LukF and LukS produce a pore with a unitary conductance of 2.5 nS (1 M KCl, 5 mM HEPES, pH 7.4), which is over three times greater than that of α-hemolysin measured under the same conditions. The subunit composition and stoichiometry of a leukocidin pore were determined by two independent methods, gel shift electrophoresis and sitespecific chemical modification during single channel recording. Four LukF and four LukS subunits were shown to co-assemble into an octameric transmembrane structure. The existence of an additional subunit in part explains properties of the leukocidin pore, such as its high conductance. Additionally, this is the first time that either technique has been applied successfully to assess the composition of a heteromeric membrane protein. It is also relevant to understanding the mechanism of assembly of β-PFT pores, and suggests new possibilities for engineering these proteins. In additional studies, the HlyII pore encoded by Bacillus cereus was found to form a homoheptameric transmembrane pore with properties conforming in general with those of other members of the class of β-PFTs. HlyII possesses additional properties which make it an attractive candidate for applications in biotechnology, such as an oligomer with a high thermal stability in the presence of SDS and the ability of the pore to remain open at high transmembrane potentials.