Stephen Cheley

Sequence-specific detection of individual DNA strands using engineered nanopores

We describe biosensor elements that are capable of identifying individual DNA strands with single-base resolution. Each biosensor element consists of an individual DNA oligonucleotide covalently attached within the lumen of the α-hemolysin (αHL) pore to form a “DNA–nanopore”. The binding of single-stranded DNA (ssDNA) molecules to the tethered DNA strand causes changes in the ionic current flowing through a nanopore. On the basis of DNA duplex lifetimes, the DNA–nanopores are able to discriminate between individual DNA strands up to 30 nucleotides in length differing by a single base substitution. This was exemplified by the detection of a drug resistance–conferring mutation in the reverse transcriptase gene of HIV. In addition, the approach was used to sequence a complete codon in an individual DNA strand tethered to a nanopore.

Stochastic sensing of organic analytes by a pore-forming protein containing a molecular adapter.

The detection of organic molecules is important in many areas, including medicine, environmental monitoring and defence. Stochastic sensing is an approach that relies on the observation of individual binding events between analyte molecules and a single receptor. Engineered transmembrane protein pores are promising sensor elements for stochastic detection, and in their simplest manifestation they produce a fluctuating binary (‘on/off’) response in the transmembrane electrical current. The frequency of occurrence of the fluctuations reveals the concentration of the analyte, and its identity can be deduced from the characteristic magnitude and/or duration of the fluctuations. Genetically engineered versions of the bacterial pore-forming protein α-haemolysin have been used to identify and quantify divalent metal ions in solution. But it is not immediately obvious how versatile binding sites for organic ligands might be obtained by engineering of the pore structure. Here we show that stochastic sensing of organic molecules can be procured from α-haemolysin by equipping the channel with an internal, non-covalently bound molecular ‘adapter’ which mediates channel blocking by the analyte. We use cyclodextrins as the adapters because these fit comfortably inside the pore and present a hydrophobic cavity suitable for binding a variety of organic analytes. Moreover, a single sensing element of this sort can be used to analyse a mixture of organic molecules with different binding characteristics. We envisage the use of other adapters, so that the pore could be ‘programmed’ for a range of sensing functions.

Intracellular trehalose improves the survival of cryopreserved mammalian cells.

We report that the introduction of low concentrations of intracellular trehalose can greatly improve the survival of mammalian cells during cryopreservation. Using a genetically engineered mutant of Staphylococcus aureus α-hemolysin to create pores in the cellular membrane, we were able to load trehalose into cells. Low concentrations (0.2 M) of trehalose permitted long-term post-thaw survival of more than 80% of 3T3 fibroblasts and 70% of human keratinocytes. These results indicate that simplified and widely applicable freezing protocols may be possible using sugars as intracellular cryoprotective additives.

Simultaneous stochastic sensing of divalent metal ions.

Stochastic sensing is an emerging analytical technique that relies upon single-molecule detection. Transmembrane pores, into which binding sites for analytes have been placed by genetic engineering, have been developed as stochastic sensing elements. Reversible occupation of an engineered binding site modulates the ionic current passing through a pore in a transmembrane potential and thereby provides both the concentration of an analyte and, through a characteristic signature, its identity. Here, we show that the concentrations of two or more divalent metal ions in solution can be determined simultaneously with a single sensor element. Further, the sensor element can be permanently calibrated without a detailed understanding of the kinetics of interaction of the metal ions with the engineered pore.

Purification and characterization of recombinant spider silk expressed in Escherichia coli

Abstract A partial cDNA clone, from the 3′ end of the dragline silk gene was isolated from Nephila clavipes major ampullate glands. This clone contains a 1.7-kb insert, consisting of a repetitive coding region of 1.4-kb and a 0.3-kb nonrepetitive coding region; 1.5-kb of the 1.7-kb fragment was cloned into Escherichia coli and a␣43-kDa recombinant silk protein was expressed. Characterization of the purified protein by Western blot, amino acid composition analysis, and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry confirms it to be spider dragline silk.

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.

An intermediate in the assembly of a pore-forming protein trapped with a genetically-engineered switch

BACKGROUND Studies of the mechanisms by which certain water-soluble proteins can assemble into lipid bilayers are relevant to several areas of biology, including the biosynthesis of membrane and secreted proteins, virus membrane fusion and the action of immune proteins such as complement and perforin. The alpha-hemolysin (alpha HL) protein, an exotoxin secreted by Staphylococcus aureus that forms heptameric pores in lipid bilayers, is a useful model for studying membrane protein assembly. In addition, modified alpha HL might be useful as a component of biosensors or in drug delivery. We have therefore used protein engineering to produce variants of alpha HL that contain molecular triggers and switches with which pore-forming activity can be modulated at will. Previously, we showed that the conductance of pores formed by the mutant hemolysin alpha HL-H5, which contains a Zn(II)-binding pentahistidine sequence, is blocked by Zn(II) from either side of the lipid bilayer, suggesting that residues from the pentahistidine sequence line the lumen of the transmembrane channel. RESULTS Here we show that Zn(II) can arrest the assembly of alpha HL-H5 before pore formation by preventing an impermeable oligomeric prepore from proceeding to the fully assembled state. The prepore is a heptamer. Limited proteolysis shows that, unlike the functional pore, the prepore contains sites near the amino terminus of the polypeptide chain that are exposed to the aqueous phase. Upon removal of the bound Zn(II) with EDTA, pore formation is completed and the sites near the amino terminus become occluded. Conversion of the prepore to the active pore is the rate-determining step in assembly and cannot be reversed by the subsequent addition of excess Zn(II). CONCLUSIONS The introduction of a simple Zn(II)-binding motif into a pore-forming protein has allowed the isolation of a defined intermediate in assembly. Genetically-engineered switches for trapping and releasing intermediates that are actuated by metal coordination or other chemistries might be generally useful for analyzing the assembly of membrane proteins and other supramolecular structures.

Stochastic sensing of TNT with a genetically engineered pore

Engineered versions of the transmembrane protein pore α‐hemolysin (αHL) 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 αHL 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.

Electroosmotic enhancement of the binding of a neutral molecule to a transmembrane pore

The flux of solvent water coupled to the transit of ions through protein pores is considerable. The effect of this electroosmotic solvent flow on the binding of a neutral molecule [β-cyclodextrin (βCD)] to sites within the staphylococcal α-hemolysin pore was investigated. Mutant α-hemolysin pores were used to which βCD can bind from either entrance and through which the direction of water flow can be controlled by choosing the charge selectivity of the pore and the polarity of the applied potential. The Kd values for βCD for individual mutant pores varied by >100-fold with the applied potential over a range of –120 to +120 mV. In all cases, the signs of the changes in binding free energy and the influence of potential on the association and dissociation rate constants for βCD were consistent with an electroosmotic effect.

Partitioning of Individual Flexible Polymers into a Nanoscopic Protein Pore

Polymer dynamics are of fundamental importance in materials science, biotechnology, and medicine. However, very little is known about the kinetics of partitioning of flexible polymer molecules into pores of nanometer dimensions. We employed electrical recording to probe the partitioning of single poly(ethylene glycol) (PEG) molecules, at concentrations near the dilute regime, into the transmembrane beta-barrel of individual protein pores formed from staphylococcal alpha-hemolysin (alphaHL). The interactions of the alpha-hemolysin pore with the PEGs (M(w) 940-6000 Da) fell into two classes: short-duration events (tau approximately 20 micro s), approximately 85% of the total, and long-duration events (tau approximately 100 micro s), approximately 15% of the total. The association rate constants (k(on)) for both classes of events were strongly dependent on polymer mass, and values of k(on) ranged over two orders of magnitude. By contrast, the dissociation rate constants (k(off)) exhibited a weak dependence on mass, suggesting that the polymer chains are largely compacted before they enter the pore, and do not decompact to a significant extent before they exit. The values of k(on) and k(off) were used to determine partition coefficients (Pi) for the PEGs between the bulk aqueous phase and the pore lumen. The low values of Pi are in keeping with a negligible interaction between the PEG chains and the interior surface of the pore, which is independent of ionic strength. For the long events, values of Pi decrease exponentially with polymer mass, according to the scaling law of Daoud and de Gennes. For PEG molecules larger than approximately 5 kDa, Pi reached a limiting value suggesting that these PEG chains cannot fit entirely into the beta-barrel.