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Biomedical subjects

Hagan Bayley

Publications and source records attributed to Hagan Bayley.

At least 19 recordsLinked to original sources
PubMed2006

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.

Azo Compounds↗
PubMed2006

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.

Hemolysin Proteins↗
PubMed2006

Direct transfer of membrane proteins from bacteria to planar bilayers for rapid screening by single-channel recording.

Although the examination of membrane proteins in planar bilayers is a powerful methodology for evaluating their pharmacology and physiological roles, introducing membrane proteins into bilayers is often a difficult process. Here, we use a mechanical probe to transfer membrane proteins directly from Escherichia coli expression colonies to artificial lipid bilayers. In this way, single-channel electrical recordings can be made from both of the major classes of membrane proteins, alpha-helix bundles and beta barrels, which are represented respectively by a K(+) channel and a bacterial pore-forming toxin. Further, we examined the bicomponent toxin leukocidin (Luk), which is composed of LukF and LukS subunits. We mixed separate LukF- and LukS-expressing colonies and transferred the mixture to a planar bilayer, which generated functional Luk pores. By this means, we rapidly screened binary combinations of mutant Luk subunits for a specific function: the ability to bind a molecular adaptor. We suggest that direct transfer from cells to bilayers will be useful in several aspects of membrane proteomics and in the construction of sensor arrays.

Bacterial Proteins↗
PubMed2006

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.

DNA↗
PubMed2006

A genetically encoded pore for the stochastic detection of a protein kinase.

Stochastic sensing is an emerging approach for the detection of a wide variety of analytes at the level of individual molecules. Detection is accomplished by observing the modulation of the current that flows through a single protein pore that has been engineered to bind an analyte of interest. Previously, protein analytes have been detected by using pores to which ligands have been appended at specific sites by targeted chemical modification. Here, we report the first genetically encoded stochastic sensor element for detecting a protein. A protein kinase inhibitor peptide sequence was incorporated into the alpha-hemolysin polypeptide, which was used to form a heteroheptameric pore containing a single copy of the inhibitor sequence. With this pore, the successful detection of the catalytic subunit of protein kinase A was demonstrated. This development should greatly facilitate the detection of active kinase subunits by stochastic sensing and the rapid screening of kinase inhibitors by an approach that yields kinetic information.

Base Sequence↗
PubMed2005

Assembly of the Bi-component leukocidin pore examined by truncation mutagenesis.

Staphylococcal leukocidin (Luk) and alpha-hemolysin (alphaHL) are members of the same family of beta barrel pore-forming toxins (betaPFTs). Although the alphaHL pore is a homoheptamer, the Luk pore is formed by the co-assembly of four copies each of the two distantly related polypeptides, LukF and LukS, to form an octamer. Here, we examine N- and C-terminal truncation mutants of LukF and LukS. LukF subunits missing up to nineteen N-terminal amino acids are capable of producing stable, functional hetero-oligomers with WT LukS. LukS subunits missing up to fourteen N-terminal amino acids perform similarly in combination with WT LukF. Further, the simultaneous truncation of both LukF and LukS is tolerated. Both Luk subunits are vulnerable to short deletions at the C terminus. Interestingly, the N terminus of the LukS polypeptide becomes resistant to proteolytic digestion in the fully assembled Luk pore while the N terminus of LukF remains in an exposed conformation. The results from this work and related experiments on alphaHL suggest that, although the N termini of betaPFTs may undergo reorganization during assembly, they are dispensable for the formation of functional pores.

Amino Acid Sequence↗
PubMed2005

Protein components for nanodevices.

A long-term goal of nanobiotechnology is to build tiny devices that respond to the environment, perform computations and carry out tasks. Considerable progress has been made in building protein components for such devices, and here we describe examples, including self-assembling protein arrays, pores with triggers and switches, and motor proteins harnessed for specific tasks. A major issue that has been successfully addressed in this recent work is the interface between the proteins and other components of the system, such as a metal surface. While further progress is expected in the coming years, the assembly of devices from the components has seen more limited accomplishments. For example, although a wide variety of sensors based on nanobiotechnology has been developed, unresolved problems still confront the construction of complex nanobioelectronic circuits, and the development of nanorobotics with biological components remains a distant dream.

Microchip Analytical Procedures↗
PubMed2005

Role of the amino latch of staphylococcal alpha-hemolysin in pore formation: a co-operative interaction between the N terminus and position 217.

Staphylococcal alpha-hemolysin (alphaHL) is a beta barrel pore-forming toxin that is secreted by the bacterium as a water-soluble monomeric protein. Upon binding to susceptible cells, alphaHL assembles via an inactive prepore to form a water-filled homoheptameric transmembrane pore. The N terminus of alphaHL, which in the crystal structure of the fully assembled pore forms a latch between adjacent subunits, has been thought to play a vital role in the prepore to pore conversion. For example, the deletion of two N-terminal residues produced a completely inactive protein that was arrested in assembly at the prepore stage. In the present study, we have re-examined assembly with a comprehensive set of truncation mutants. Surprisingly, we found that after truncation of up to 17 amino acids, the ability of alphaHL to form functional pores was diminished, but still substantial. We then discovered that the mutation Ser(217) --> Asn, which was present in our original set of truncations but not in the new ones, promotes complete inactivation upon truncation of the N terminus. Therefore, the N terminus of alphaHL cannot be critical for the prepore to pore transformation as previously thought. Residue 217 is involved in the assembly process and must interact indirectly with the distant N terminus during the last step in pore formation. In addition, we provide evidence that an intact N terminus prevents the premature oligomerization of alphaHL monomers in solution.

Amino Acid Sequence↗
PubMed2005

Stepwise growth of a single polymer chain.

By monitoring the modulation of an ionic current passing through a nanoreactor formed from a protein pore, the step-by-step growth of an individual polymer chain was monitored. The observation of polymer growth at the single-molecule level will be useful for studying the kinetics of chain growth or the movement of polymers under confinement. It might also be used to synthesize "molecular fishing lines" in situ, for applications in stochastic sensing.

Journal Article↗
PubMed2005

The internal cavity of the staphylococcal alpha-hemolysin pore accommodates approximately 175 exogenous amino acid residues.

The cavity within the cap domain of the transmembrane staphylococcal alpha-hemolysin (alphaHL) pore is roughly a sphere of diameter approximately 45 A (molecular surface volume approximately 39,500 A(3)). We tested the ability of the cavity to accommodate exogenous polypeptide chains. Concatemerized Gly/Ser-containing sequences ("loops", L; number of repeats = n; number of residues = 10n + 5, n = 0-21) were inserted at a position located within the cavity of the fully assembled heptameric alphaHL pore. Homomeric pores containing 25 or less residues in each loop (n or= 7, only one L subunit was incorporated. As the inserted loop was lengthened, transient closures were observed in planar bilayer experiments with single pores. However, L(1)W(6) pores with very long loops (n = 14 and 21) had unitary conductance values close to those of W(7), suggesting that the loop is extruded through the opening in the cap of the pore into the external medium. Further analysis of bilayer recordings and electrophoretic migration patterns indicates that the upper capacity of the cavity is approximately 175 amino acids. The findings suggest that small functional peptides or proteins might be assembled within the alphaHL pore.

Amino Acids↗
PubMed2005

Interactions of peptides with a protein pore.

The partitioning of polypeptides into nanoscale transmembrane pores is of fundamental importance in biology. Examples include protein translocation in the endoplasmic reticulum and the passage of proteins through the nuclear pore complex. Here we examine the exchange of cationic alpha-helical peptides between the bulk aqueous phase and the transmembrane beta-barrel of the alpha-hemolysin (alphaHL) protein pore at the single-molecule level. The experimental kinetic data suggest a two-barrier, single-well free energy profile for peptide transit through the alphaHL pore. This free energy profile is strongly voltage- and peptide-length-dependent. We used the Woodhull-Eyring formalism to rationalize the values measured for the association and dissociation rate constants k(on) and k(off), and to separate k(off) into individual rate constants for exit through each of the openings of the protein pore. The rate constants k(on), k(off)(cis), and k(off)(trans) decreased with the length of the peptide. At high transmembrane potentials, the alanine-based peptides, which include bulky lysine side chains, bind more strongly (formation constants K(f) approximately tens of M(-1)) than highly flexible polyethylene glycols (K(f) approximately M(-1)) to the lumen of the alphaHL protein pore. In contrast, at zero transmembrane potential, the peptides bind weakly to the lumen of the pore, and the affinity decreases with the peptide length, similar to the case of the polyethylene glycols. The binding is enhanced at increased transmembrane potentials, because the free energy contribution DeltaG = -zetadeltaFV/RT predominates with the peptides. We suggest that the alphaHL protein may serve as a robust and versatile model for examining the interactions between positively charged signal peptides and a beta-barrel pore.

Bacterial Toxins↗
PubMed2005

Direct introduction of single protein channels and pores into lipid bilayers.

We have developed a mechanical method for inserting single pores and channels into lipid bilayers. A hand-operated hydrogel probe, coated with a layer of proteins, is mechanically engaged with the lipid bilayer. The two major classes of membrane proteins (beta barrels and alpha-helix bundles) that can be inserted, thereby demonstrating the wide applicability of the approach. Recordings from the proteins show that they retain electrical properties that are the same as those of proteins inserted from solution. Protein-loaded probes can be used repeatedly, allowing individual pores to be rapidly screened one at a time. The method has implications for fundamental studies of cell membranes, array fabrication, and chemical screening.

Bacterial Proteins↗
PubMed2005

Stochastic sensing of TNT with a genetically engineered pore.

Engineered versions of the transmembrane protein pore alpha-hemolysin (alphaHL) can be used as stochastic sensing elements for the identification and quantification of a wide variety of analytes at the single-molecule level. Until now, nitroaromatic analytes have eluded detection by this approach. We now report that binding sites for nitroaromatics can be built within the lumen of the alphaHL pore from simple rings of seven aromatic amino acid side chains (Phe, Tyr or Trp). By monitoring the ionic current that passes through a single pore at a fixed applied potential, various nitroaromatics can be distinguished from TNT on the basis of the amplitude and duration of individual current-blocking events. Rings of less than seven aromatics bind the analytes more weakly; this suggests that direct aromatic-aromatic interactions are involved. The engineered pores should be useful for the detection of explosives and, in combination with computational approaches and structural analysis, they could further our understanding of noncovalent interactions between aromatic molecules.

Biosensing Techniques↗
PubMed2005

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.

Adenosine Triphosphate↗