Prolonged residence time of a noncovalent molecular adapter, beta-cyclodextrin, within the lumen of mutant alpha-hemolysin pores.

Noncovalent molecular adapters, such as cyclodextrins, act as binding sites for channel blockers when lodged in the lumen of the alpha-hemolysin (alphaHL) pore, thereby offering a basis for the detection of a variety of organic molecules with alphaHL as a sensor element. beta-Cyclodextrin (betaCD) resides in the wild-type alphaHL pore for several hundred microseconds. The residence time can be extended to several milliseconds by the manipulation of pH and transmembrane potential. Here, we describe mutant homoheptameric alphaHL pores that are capable of accommodating betaCD for tens of seconds. The mutants were obtained by site-directed mutagenesis at position 113, which is a residue that lies near a constriction in the lumen of the transmembrane beta barrel, and fall into two classes. Members of the tight-binding class, M113D, M113N, M113V, M113H, M113F and M113Y, bind betaCD approximately 10(4)-fold more avidly than the remaining alphaHL pores, including WT-alphaHL. The lower K(d) values of these mutants are dominated by reduced values of k(off). The major effect of the mutations is most likely a remodeling of the binding site for betaCD in the vicinity of position 113. In addition, there is a smaller voltage-sensitive component of the binding, which is also affected by the residue at 113 and may result from transport of the neutral betaCD molecule by electroosmotic flow. The mutant pores for which the dwell time of betaCD is prolonged can serve as improved components for stochastic sensors.

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 alpha-hemolysin (alphaHL) 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.

The staphylococcal leukocidin bicomponent toxin forms large ionic channels.

The genes encoding the F and S components of a leukocidin, LukF (HlgB) and LukS (HlgC), a pore-forming binary toxin, were amplified from the Smith 5R strain of Staphylococcus aureus both with and without sequences encoding 3'-hexahistidine tags. The His-tagged components were expressed in Escherichia coli and purified under nondenaturing conditions. In addition, the two unmodified proteins and the His-tagged versions were produced in an E. coli cell-free in vitro transcription and translation system. An SDS-stable oligomer of approximately 200 kDa appeared when both components were cotranslated in the presence of rabbit erythrocyte membranes. Hemolytic activity of the combined components against rabbit erythrocytes was measured for both in vitro- and in vivo-produced polypeptides, yielding similar HC(50) values of approximately 0.14 microg/mL. The pore-forming properties of the recombinant leukocidin were also investigated with planar lipid bilayers of diphytanoylphosphatidylcholine. Although leukocidins and staphylococcal alpha-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 and 5 mM HEPES (pH 7.4)], which is more than 3 times greater than that of alpha-hemolysin measured under the same conditions. Therefore, if the leukocidin pore were a cylinder, its diameter would be almost twice that of alpha-hemolysin. In addition, the leukocidin pore is weakly cation selective and exhibits gating at low positive potentials, while alpha-hemolysin is weakly anion selective and gates only at high potentials. Taken together, these data suggest that the structure of the oligomeric pore formed by the leukocidin examined here has diverged significantly from that of alpha-hemolysin.

Location of a constriction in the lumen of a transmembrane pore by targeted covalent attachment of polymer molecules.

Few methods exist for obtaining the internal dimensions of transmembrane pores for which 3-D structures are lacking or for showing that structures determined by crystallography reflect the internal dimensions of pores in lipid bilayers. Several approaches, involving polymer penetration and transport, have revealed limiting diameters for various pores. But, in general, these approaches do not indicate the locations of constrictions in the channel lumen. Here, we combine cysteine mutagenesis and chemical modification with sulfhydryl-reactive polymers to locate the constriction in the lumen of the staphylococcal alpha-hemolysin pore, a model protein of known structure. The rates of reaction of each of four polymeric reagents (MePEG-OPSS) of different masses towards individual single cysteine mutants, comprising a set with cysteines distributed over the length of the lumen of the pore, were determined by macroscopic current recording. The rates for the three larger polymers (1.8, 2.5, and 5.0 kD) were normalized with respect to the rates of reaction with a 1.0-kD polymer for each of the seven positions in the lumen. The rate of reaction of the 5.0-kD polymer dropped dramatically at the centrally located Cys-111 residue and positions distal to Cys-111, whether the reagent was applied from the trans or the cis side of the bilayer. This semi-quantitative analysis sufficed to demonstrate that a constriction is located at the midpoint of the pore lumen, as predicted by the crystal structure, and although the constriction allows a 2.5-kD polymer to pass, transport of a 5.0-kD molecule is greatly restricted. In addition, PEG chains gave greater reductions in pore conductance when covalently attached to the narrower regions of the lumen, permitting further definition of the interior of the pore. The procedures described here should be applicable to other pores and to related structures such as the vestibules of ion channels.

Capture of a single molecule in a nanocavity.

We describe a heptameric protein pore that has been engineered to accommodate two different cyclodextrin adapters simultaneously within the lumen of a transmembrane beta barrel. The volume between the adapters is a cavity of approximately 4400 cubic angstroms. Analysis of single-channel recordings reveals that individual charged organic molecules can be pulled into the cavity by an electrical potential. Once trapped, an organic molecule shuttles back and forth between the adapters for hundreds of milliseconds. Such self-assembling nanostructures are of interest for the fabrication of multianalyte sensors and could provide a means to control chemical reactions.

Beneficial effect of intracellular trehalose on the membrane integrity of dried mammalian cells.

Recently, there has been much interest in using trehalose and other small carbohydrates to preserve mammalian cells in the dried state as an alternative to cryopreservation. Here, we report on the successful preservation of plasma membrane integrity after drying, as a first step toward full preservation of mammalian cells. Trehalose was introduced into cells using a genetically engineered version of alpha-hemolysin, a pore-forming protein; the cells were then dried and stored for weeks at different temperatures with approximately 90% recovery of the intact plasma membrane. We show that protection of the plasma membrane by internal trehalose is dose dependent and estimate the amount of internal trehalose required for adequate protection to be approximately 10(10) molecules/cell. In addition, a minimal amount of water (approximately 15 wt%) appears to be necessary. These results show that a key component of mammalian cells can be preserved in a dried state for weeks under mild conditions (-20 degrees C and 5% relative humidity) and thereby suggest new approaches to preserving mammalian cells.

Biochemical and biophysical characterization of OmpG: A monomeric porin.

A recombinant form of the porin OmpG, OmpGm, lacking the signal sequence, has been expressed in Escherichia coli. After purification under denaturing conditions, the protein was refolded in the detergent Genapol X-080, where it gained a structure rich in beta sheet as evidenced by a CD spectrum similar to that of the native form. Electrophoretic analysis and limited proteolysis experiments suggested that refolded OmpGm exists in at least three forms. Nevertheless, the recombinant protein formed uniform channels in planar bilayers with a conductance of 0.81 nS (1 M NaCl, pH 7.5). Previous biochemical studies had suggested that OmpG is a monomeric porin, rather than the usual trimer. Bilayer recordings substantiated this proposal; voltage-induced closures occurred consistently in a single step, and channel block by Gd(3+) lacked the cooperativity seen with the trimeric porin OmpF. The availability of milligram amounts of a monomeric porin will be useful both for basic studies of porin function and for membrane protein engineering.

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.

Reversal of charge selectivity in transmembrane protein pores by using noncovalent molecular adapters.

In this study, the charge selectivity of staphylococcal alpha-hemolysin (alphaHL), a bacterial pore-forming toxin, is manipulated by using cyclodextrins as noncovalent molecular adapters. Anion-selective versions of alphaHL, including the wild-type pore and various mutants, become more anion selective when beta-cyclodextrin (betaCD) is lodged within the channel lumen. By contrast, the negatively charged adapter, hepta-6-sulfato-beta-cyclodextrin (s(7)betaCD), produces cation selectivity. The cyclodextrin adapters have similar effects when placed in cation-selective mutant alphaHL pores. Most probably, hydrated Cl(-) ions partition into the central cavity of betaCD more readily than K(+) ions, whereas s(7)betaCD introduces a charged ring near the midpoint of the channel lumen and confers cation selectivity through electrostatic interactions. The molecular adapters generate permeability ratios (P(K+)/P(Cl-)) over a 200-fold range and should be useful in the de novo design of membrane channels both for basic studies of ion permeation and for applications in biotechnology.

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 alpha-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.

A functional protein pore with a "retro" transmembrane domain.

Extended retro (reversed) peptide sequences have not previously been accommodated within functional proteins. Here, we show that the entire transmembrane portion of the beta-barrel of the pore-forming protein alpha-hemolysin can be formed by retrosequences comprising a total of 175 amino acid residues, 25 contributed by the central sequence of each subunit of the heptameric pore. The properties of wild-type and retro heptamers in planar bilayers are similar. The single-channel conductance of the retro pore is 15% less than that of the wild-type heptamer and its current-voltage relationship denotes close to ohmic behavior, while the wild-type pore is weakly rectifying. Both wild-type and retro pores are very weakly anion selective. These results and the examination of molecular models suggest that beta-barrels may be especially accepting of retro sequences compared to other protein folds. Indeed, the ability to form a retro domain could be diagnostic of a beta-barrel, explaining, for example, the activity of the retro forms of many membrane-permeabilizing peptides. By contrast with the wild-type subunits, monomeric retro subunits undergo premature assembly in the absence of membranes, most likely because the altered central sequence fails to interact with the remainder of the subunit, thereby initiating assembly. Despite this difficulty, a technique was devised for obtaining heteromeric pores containing both wild-type and retro subunits. Most probably as a consequence of unfavorable interstrand side-chain interactions, the heteromeric pores are less stable than either the wild-type or retro homoheptamers, as judged by the presence of subconductance states in single-channel recordings. Knowledge about the extraordinary plasticity of the transmembrane beta-barrel of alpha-hemolysin will be very useful in the de novo design of functional membrane proteins based on the beta-barrel motif.

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 alpha-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 alpha-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.

Heptameric structures of two alpha-hemolysin mutants imaged with in situ atomic force microscopy.

Atomic force microscopy has been used to study self-assembled structures of two alpha-hemolysin mutants. For a mutant (alphaHL-H5) that was locked into the prepore state on fluid phase egg-PC membranes, we visualized, for the first time, heptameric prepores and showed that the 7-fold axis in the prepore lies perpendicular to the membrane surface. For another mutant (TCM) with the transmembrane domain, the self-assembled oligomer that assumes the conformation of the fully assembled pore is also a heptamer. These results show that heptamers are the preferred oligomerization state of alpha-hemolysin.

Genetically engineered metal ion binding sites on the outside of a Channel's transmembrane beta-barrel.

We are exploring the ability of genetically engineered versions of the Staphylococcus aureus alpha-hemolysin (alphaHL) ion channel to serve as rationally designed sensor components for analytes including divalent cations. We show here that neither the hemolytic activity nor the single channel current of wild-type alphaHL was affected by [Zn(II)]

Purification and characterization of recombinant spider silk expressed in Escherichia coli.

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.

The heptameric prepore of a staphylococcal alpha-hemolysin mutant in lipid bilayers imaged by atomic force microscopy.

We have used atomic force microscopy to study the oligomeric state of a genetically engineered mutant of staphylococcal alpha-hemolysin (alphaHL-H5) that can be arrested as a "prepore" assembly intermediate. AFM images of alphaHL-H5 on supported bilayers of a fluid-phase lipid, egg-yolk phosphatidylcholine (egg-PC), under conditions that lock alphaHL-H5 into the prepore state, clearly show a heptameric structure for many individual oligomers. The central dent of the prepore has a diameter of 3.2 +/- 0.2 nm. The distance between the centers of mass of neighboring subunits is 2.8 +/- 0.3 nm. The heptamer has an average diameter of 8.9 +/- 0.6 nm. These results support a recently proposed pathway for the assembly of alpha-hemolysin.

Designed protein pores as components for biosensors.

BACKGROUND: There is a pressing need for new sensors that can detect a variety of analytes, ranging from simple ions to complex compounds and even microorganisms. The devices should offer sensitivity, speed, reversibility and selectivity. Given these criteria, protein pores, remodeled so that their transmembrane conductances are modulated by the association of specific analytes, are excellent prospects as components of biosensors. RESULTS: Structure-based design and a separation method that employs targeted chemical modification have been used to obtain a heteromeric form of the bacterial pore-forming protein staphylococcal alpha-hemolysin, in which one of the seven subunits contains a binding site for a divalent metal ion, M(II), which serves as a prototypic analyte. The single-channel current of the heteromer in planar bilayers is modulated by nanomolar Zn(II). Other M(II)s modulate the current and produce characteristic signatures. In addition, heteromers containing more than one mutant subunit exhibit distinct responses to M(II)s Hence, a large collection of responsive pores can be generated through subunit diversity and combinatorial assembly. CONCLUSIONS: Engineered pores have several advantages as potential sensor elements: sensitivity is in the nanomolar range; analyte binding is rapid (diffusion limited in some cases) and reversible; strictly selective binding is not required because single-channel recordings are rich in information; and for a particular analyte, the dissociation rate constant, the extent of channel block and the voltage-dependence of these parameters are distinguishing, while the frequency of partial channel block reflects the analyte concentration. A single sensor element might, therefore, be used to quantitate more than one analyte at once. The approach described here can be generalized for additional analytes.

Spontaneous oligomerization of a staphylococcal alpha-hemolysin conformationally constrained by removal of residues that form the transmembrane beta-barrel.

Staphylococcal alpha-hemolysin is a water soluble, monomeric, bacterial exotoxin, which forms heptameric pores in membranes. The rate determining step in assembly is the conversion of a heptameric prepore to the fully assembled pore in which the central glycine-rich domain of each subunit inserts into the membrane to form a 14 strand beta barrel. Barrel formation is accompanied by a conformational change in which each N terminus latches onto an adjacent subunit. In the monomer in solution, the central domain is loosely organized and exposed to solvent. In this study, 25 amino acids of the central domain were removed and replaced with the sequence Asp-Gly, which favors the formation of a type I' beta-turn, to yield a mutant devoid of hemolytic activity. Within minutes after synthesis in the absence of membranes, the mutant polypeptide spontaneously assembled into heptamers, as demonstrated by atomic force microscopy. Limited proteolysis suggested that the N termini of the subunits in the heptamers were in the fully assembled pore conformation rather than the prepore conformation. Based on these findings, the deletion is proposed to constrain the central domain and thereby force the creation of a shortened beta barrel, which in turn induces the additional structural changes that normally accompany pore formation. The truncated pore might make a useful framework for the construction of designed membrane active macromolecules.

Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore.

The structure of the Staphylococcus aureus alpha-hemolysin pore has been determined to 1.9 A resolution. Contained within the mushroom-shaped homo-oligomeric heptamer is a solvent-filled channel, 100 A in length, that runs along the sevenfold axis and ranges from 14 A to 46 A in diameter. The lytic, transmembrane domain comprises the lower half of a 14-strand antiparallel beta barrel, to which each protomer contributes two beta strands, each 65 A long. The interior of the beta barrel is primarily hydrophilic, and the exterior has a hydrophobic belt 28 A wide. The structure proves the heptameric subunit stoichiometry of the alpha-hemolysin oligomer, shows that a glycine-rich and solvent-exposed region of a water-soluble protein can self-assemble to form a transmembrane pore of defined structure, and provides insight into the principles of membrane interaction and transport activity of beta barrel pore-forming toxins.

Tumor protease-activated, pore-forming toxins from a combinatorial library.

We describe a library of two-chain molecular complementation mutants of staphylococcal alpha-hemolysin that features a combinatorial cassette encoding thousands of protease recognition sites in the central pore-forming domain. The cassette is flanked by a peptide extension that inactivates the protein. We screened the library to identify alpha-hemolysins that are highly susceptible to activation by cathepsin B, a protease that is secreted by certain metastatic tumor cells. Toxins obtained by this procedure should be useful for the permeabilization of malignant cells thereby leading directly to cell death or permitting destruction of the cells with drugs that are normally membrane impermeant.