Stochastic detection of Pim protein kinases reveals electrostatically enhanced association of a peptide substrate.

In stochastic sensing, the association and dissociation of analyte molecules is observed as the modulation of an ionic current flowing through a single engineered protein pore, enabling the label-free determination of rate and equilibrium constants with respect to a specific binding site. We engineered sensors based on the staphylococcal α-hemolysin pore to allow the single-molecule detection and characterization of protein kinase-peptide interactions. We enhanced this approach by using site-specific proteolysis to generate pores bearing a single peptide sensor element attached by an N-terminal peptide bond to the trans mouth of the pore. Kinetics and affinities for the Pim protein kinases (Pim-1, Pim-2, and Pim-3) and cAMP-dependent protein kinase were measured and found to be independent of membrane potential and in good agreement with previously reported data. Kinase binding exhibited a distinct current noise behavior that forms a basis for analyte discrimination. Finally, we observed unusually high association rate constants for the interaction of Pim kinases with their consensus substrate Pimtide (~10(7) to 10(8) M(-1) · s(-1)), the result of electrostatic enhancement, and propose a cellular role for this phenomenon.

Autocrine activation of P2Y1 receptors couples Ca (2+) influx to Ca (2+) release in human pancreatic beta cells.

AIMS/HYPOTHESIS: There is evidence that ATP acts as an autocrine signal in beta cells but the receptors and pathways involved are incompletely understood. Here we investigate the receptor subtype(s) and mechanism(s) mediating the effects of ATP on human beta cells. METHODS: We examined the effects of purinergic agonists and antagonists on membrane potential, membrane currents, intracellular Ca(2+) ([Ca(2+)]i) and insulin secretion in human beta cells. RESULTS: Extracellular application of ATP evoked small inward currents (3.4 ± 0.7 pA) accompanied by depolarisation of the membrane potential (by 14.4 ± 2.4 mV) and stimulation of electrical activity at 6 mmol/l glucose. ATP increased [Ca(2+)]i by stimulating Ca(2+) influx and evoking Ca(2+) release via InsP3-receptors in the endoplasmic reticulum (ER). ATP-evoked Ca(2+) release was sufficient to trigger exocytosis in cells voltage-clamped at -70 mV. All effects of ATP were mimicked by the P2Y(1/12/13) agonist ADP and the P2Y1 agonist MRS-2365, whereas the P2X(1/3) agonist α,ß-methyleneadenosine-5-triphosphate only had a small effect. The P2Y1 antagonists MRS-2279 and MRS-2500 hyperpolarised glucose-stimulated beta cells and lowered [Ca(2+)]i in the absence of exogenously added ATP and inhibited glucose-induced insulin secretion by 35%. In voltage-clamped cells subjected to action potential-like stimulation, MRS-2279 decreased [Ca(2+)]i and exocytosis without affecting Ca(2+) influx. CONCLUSIONS/INTERPRETATION: These data demonstrate that ATP acts as a positive autocrine signal in human beta cells by activating P2Y1 receptors, stimulating electrical activity and coupling Ca(2+) influx to Ca(2+) release from ER stores.

Molecular bases of cyclodextrin adapter interactions with engineered protein nanopores.

Engineered protein pores have several potential applications in biotechnology: as sensor elements in stochastic detection and ultrarapid DNA sequencing, as nanoreactors to observe single-molecule chemistry, and in the construction of nano- and micro-devices. One important class of pores contains molecular adapters, which provide internal binding sites for small molecules. Mutants of the alpha-hemolysin (alphaHL) pore that bind the adapter beta-cyclodextrin (betaCD) approximately 10(4) times more tightly than the wild type have been obtained. We now use single-channel electrical recording, protein engineering including unnatural amino acid mutagenesis, and high-resolution x-ray crystallography to provide definitive structural information on these engineered protein nanopores in unparalleled detail.

An engineered ClyA nanopore detects folded target proteins by selective external association and pore entry.

Nanopores have been used in label-free single-molecule studies, including investigations of chemical reactions, nucleic acid analysis, and applications in sensing. Biological nanopores generally perform better than artificial nanopores as sensors, but they have disadvantages including a fixed diameter. Here we introduce a biological nanopore ClyA that is wide enough to sample and distinguish large analyte proteins, which enter the pore lumen. Remarkably, human and bovine thrombins, despite 86% sequence identity, elicit characteristic ionic current blockades, which at -50 mV differ in their main current levels by 26 ± 1 pA. The use of DNA aptamers or hirudin as ligands further distinguished the protein analytes. Finally, we constructed ClyA nanopores decorated with covalently attached aptamers. These nanopores selectively captured and internalized cognate protein analytes but excluded noncognate analytes, in a process that resembles transport by nuclear pores.

SSTR2 is the functionally dominant somatostatin receptor in human pancreatic ß- and α-cells.

Somatostatin-14 (SST) inhibits insulin and glucagon secretion by activating G protein-coupled somatostatin receptors (SSTRs), of which five isoforms exist (SSTR1-5). In mice, the effects on pancreatic ß-cells are mediated by SSTR5, whereas α-cells express SSTR2. In both cell types, SSTR activation results in membrane hyperpolarization and suppression of exocytosis. Here, we examined the mechanisms by which SST inhibits secretion from human ß- and α-cells and the SSTR isoforms mediating these effects. Quantitative PCR revealed high expression of SSTR2, with lower levels of SSTR1, SSTR3, and SSTR5, in human islets. Immunohistochemistry showed expression of SSTR2 in both ß- and α-cells. SST application hyperpolarized human ß-cells and inhibited action potential firing. The membrane hyperpolarization was unaffected by tolbutamide but antagonized by tertiapin-Q, a blocker of G protein-gated inwardly rectifying K⁺ channels (GIRK). The effect of SST was mimicked by an SSTR2-selective agonist, whereas a SSTR5 agonist was marginally effective. SST strongly (>70%) reduced depolarization-evoked exocytosis in both ß- and α-cells. A slightly weaker inhibition was observed in both cell types after SSTR2 activation. SSTR3- and SSTR1-selective agonists moderately reduced the exocytotic responses in ß- and α-cells, respectively, whereas SSTR4- and SSTR5-specific agonists were ineffective. SST also reduced voltage-gated P/Q-type Ca²âº currents in ß-cells, but normalization of Ca²âº influx to control levels by prolonged depolarizations only partially restored exocytosis. We conclude that SST inhibits secretion from both human ß- and α-cells by activating GIRK and suppressing electrical activity, reducing P/Q-type Ca²âº currents, and directly inhibiting exocytosis. These effects are mediated predominantly by SSTR2 in both cell types.

Permeation of styryl dyes through nanometer-scale pores in membranes.

Styryl dyes are widely used to study synaptic vesicle (SV) recycling in neurons; vesicles are loaded with dye during endocytosis, and dye is subsequently released via exocytosis. During putative kiss-and-run exocytosis, efflux of dye from individual SVs has been proposed to occur via two sequential steps: dissociation from the membrane followed by permeation through a small fusion pore. To improve our understanding of the kinetics of efflux of dye from vesicles during kiss-and-run events, we examined the rates of efflux of different dyes through nanometer-scale pores formed in membranes by the toxins melittin and α-hemolysin; these pores approximate the size of fusion pores measured in neuroendocrine cells. We found that the axial diameter of each dye was a crucial determinant for permeation. Moreover, the two dyes with the largest cross-sectional areas were completely unable to pass through pores formed by a mutant α-hemolysin that has a slightly smaller pore than the wild-type toxin. The overall time constant for efflux (seconds) of each dye was orders of magnitude slower than the time constant for dissociation from membranes (milliseconds). Thus, the permeation step is rate-limiting, and this observation was further supported by atomistic molecular dynamics simulations. Together, the data reported here help provide a framework for interpreting dye destaining rates from secretory vesicles.

DNA strands from denatured duplexes are translocated through engineered protein nanopores at alkaline pH.

Nanopores are under development for the detection of a variety of analytes and the investigation of chemical reactions at the single molecule level. In particular, the analysis of nucleic acid molecules is under intense investigation, including the development of systems for rapid, low-cost DNA sequencing. Here, we show that DNA can be translocated through an engineered alphaHL protein pore at pH 11.7, a value at which dsDNA is denatured. Therefore, the alphaHL pore is sufficiently stable to entertain the possibility of direct nanopore sequencing of genomic dsDNA samples, which are more readily obtained and handled than ssDNA.

Redirecting Pore Assembly of Staphylococcal α-Hemolysin by Protein Engineering.

α-Hemolysin (αHL), a ß-barrel pore-forming toxin (ßPFT), is secreted as a water-soluble monomer by Staphylococcus aureus. Upon binding to receptors on target cell membranes, αHL assembles to form heptameric membrane-spanning pores. We have previously engineered αHL to create a protease-activatable toxin that is activated by site-specific proteolysis including by tumor proteases. In this study, we redesigned αHL so that it requires 2-fold activation on target cells through (i) binding to specific receptors, and (ii) extracellular proteolytic cleavage. To assess our strategy, we constructed a fusion protein of αHL with galectin-1 (αHLG1, αHL-Galectin-1 chimera). αHLG1 was cytolytic toward cells that lack a receptor for wild-type αHL. We then constructed protease-activatable mutants of αHLG1 (PAMαHLG1s). PAMαHLG1s were activated by matrix metalloproteinase 2 (MMP-2) and had approximately 50-fold higher cytolytic activity toward MMP-2 overexpressing cells (HT-1080 cells) than toward non-overexpressing cells (HL-60 cells). Our approach provides a novel strategy for tailoring pore-forming toxins for therapeutic applications.

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.

Semisynthetic Nanoreactor for Reversible Single-Molecule Covalent Chemistry.

Protein engineering has been used to remodel pores for applications in biotechnology. For example, the heptameric α-hemolysin pore (αHL) has been engineered to form a nanoreactor to study covalent chemistry at the single-molecule level. Previous work has been confined largely to the chemistry of cysteine side chains or, in one instance, to an irreversible reaction of an unnatural amino acid side chain bearing a terminal alkyne. Here, we present four different αHL pores obtained by coupling either two or three fragments by native chemical ligation (NCL). The synthetic αHL monomers were folded and incorporated into heptameric pores. The functionality of the pores was validated by hemolysis assays and by single-channel current recording. By using NCL to introduce a ketone amino acid, the nanoreactor approach was extended to an investigation of reversible covalent chemistry on an unnatural side chain at the single-molecule level.

A Glycine-Insulin Autocrine Feedback Loop Enhances Insulin Secretion From Human ß-Cells and Is Impaired in Type 2 Diabetes.

The secretion of insulin from pancreatic islet ß-cells is critical for glucose homeostasis. Disrupted insulin secretion underlies almost all forms of diabetes, including the most common form, type 2 diabetes (T2D). The control of insulin secretion is complex and affected by circulating nutrients, neuronal inputs, and local signaling. In the current study, we examined the contribution of glycine, an amino acid and neurotransmitter that activates ligand-gated Cl(-) currents, to insulin secretion from islets of human donors with and without T2D. We find that human islet ß-cells express glycine receptors (GlyR), notably the GlyRα1 subunit, and the glycine transporter (GlyT) isoforms GlyT1 and GlyT2. ß-Cells exhibit significant glycine-induced Cl(-) currents that promote membrane depolarization, Ca(2+) entry, and insulin secretion from ß-cells from donors without T2D. However, GlyRα1 expression and glycine-induced currents are reduced in ß-cells from donors with T2D. Glycine is actively cleared by the GlyT expressed within ß-cells, which store and release glycine that acts in an autocrine manner. Finally, a significant positive relationship exists between insulin and GlyR, because insulin enhances the glycine-activated current in a phosphoinositide 3-kinase-dependent manner, a positive feedback loop that we find is completely lost in ß-cells from donors with T2D.

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.

Properties of Bacillus cereus hemolysin II: a heptameric transmembrane pore.

The gene encoding hemolysin II (HlyII) was amplified from Bacillus cereus genomic DNA and a truncated mutant, HlyII(DeltaCT), was constructed lacking the 94 amino acid extension at the C terminus. The proteins were produced in an E. coli cell-free in vitro transcription and translation system, and were shown to assemble into SDS-stable oligomers on rabbit erythrocyte membranes and liposomes. The hemolytic activity of HlyII was measured with rabbit erythrocytes yielding an HC(50) value of 1.64 ng mL(-1), which is over 15 times more potent than staphylococcal alpha-hemolysin. HlyII(DeltaCT) was about eight times less potent than HlyII in this assay. Limited proteolysis of the oligomers formed by HlyII and HlyII(DeltaCT) on red cell membranes showed that the C-terminal extension is sensitive to digestion, while HlyII(DeltaCT) is protease resistant and migrates with an electrophoretic mobility similar to that of digested HlyII. HlyII forms moderately anion selective, rectifying pores (I(+80)/I(-80) = 0.57, 1 M KCl, pH 7.4) in planar lipid bilayers of diphytanoylphosphatidylcholine with a unitary conductance of 637 pS (1 M KCl, 5 mM HEPES, pH 7.4) and exhibits no gating over a wide range of applied potentials (-160 to +160 mV). In addition, it was demonstrated that HlyII forms a homoheptameric pore by using gel shift electrophoresis aided by a genetically encoded oligoaspartate tag. Although they share limited primary sequence identity (30%), these data confirm that HlyII is a structural and functional homolog of staphylococcal alpha-hemolysin.

Single-molecule interrogation of a bacterial sugar transporter allows the discovery of an extracellular inhibitor.

Capsular polysaccharides form the outermost protective layer around many Gram-negative bacteria. Antibiotics aimed directly at weakening this layer are not yet available. In pathogenic Escherichia coli E69, a protein, Wza, forms a pore in the outer membrane that transports K30 capsular polysaccharide from its site of synthesis to the outside of the cell. This therefore represents a prospective antibiotic target. Here we test a variety of grommet-like mimics of K30 capsular polysaccharide on wild-type Wza and on mutant open forms of the pore by electrical recording in planar lipid bilayers. The most effective glycomimetic was the unnatural cyclic octasaccharide octakis(6-deoxy-6-amino)cyclomaltooctaose (am8γCD), which blocks the α-helix barrel of Wza, a site that is directly accessible from the external medium. This glycomimetic inhibited K30 polysaccharide transport in live E. coli E69. With the protective outer membrane disrupted, the bacteria can be recognized and killed by the human immune system.

An engineered dimeric protein pore that spans adjacent lipid bilayers.

The bottom-up construction of artificial tissues is an underexplored area of synthetic biology. An important challenge is communication between constituent compartments of the engineered tissue, and between the engineered tissue and additional compartments, including extracellular fluids, further engineered tissue and living cells. Here we present a dimeric transmembrane pore that can span two adjacent lipid bilayers, and thereby allow aqueous compartments to communicate. Two heptameric staphylococcal α-hemolysin pores were covalently linked in an aligned cap-to-cap orientation. The structure of the dimer, (α7)2, was confirmed by biochemical analysis, transmission electron microscopy and single-channel electrical recording. We show that one of two ß-barrels of (α7)2 can insert into the lipid bilayer of a small unilamellar vesicle, while the other spans a planar lipid bilayer. The (α7)2 pores spanning two bilayers were also observed by transmission electron microscopy.

Wrestling with native chemical ligation.

An improved method for the semisynthesis of a potassium channel involving native chemical ligation allows the introduction of short sequences containing non-canonical amino acids at any position within the polypeptide chain. The work enhances the technology available for a range of fundamental investigations of membrane proteins and for applications of membrane channels and pores in biotechnology.

Droplet networks with incorporated protein diodes show collective properties.

Recently, we demonstrated that submicrolitre aqueous droplets submerged in an apolar liquid containing lipid can be tightly connected by means of lipid bilayers to form networks. Droplet interface bilayers have been used for rapid screening of membrane proteins and to form asymmetric bilayers with which to examine the fundamental properties of channels and pores. Networks, meanwhile, have been used to form microscale batteries and to detect light. Here, we develop an engineered protein pore with diode-like properties that can be incorporated into droplet interface bilayers in droplet networks to form devices with electrical properties including those of a current limiter, a half-wave rectifier and a full-wave rectifier. The droplet approach, which uses unsophisticated components (oil, lipid, salt water and a simple pore), can therefore be used to create multidroplet networks with collective properties that cannot be produced by droplet pairs.