Bacterial Toxin Alpha-Hemolysin
Alpha-Hemolysin: Self-Assembling Transmembrane Pore
In its fight for resources, bacterium Staphylococcus aureus secretes alpha-hemolysin monomers that bind to the outer membrane of susceptible cells. Upon binding, the monomers oligomerize to form a water-filled transmembrane channel that facilitates uncontrolled permeation of water, ions, and small organic molecules. Rapid discharge of vital molecules, such as ATP, dissipation of the membrane potential and ionic gradients, and irreversible osmotic swelling leading to the cell wall rupture (lysis), can cause death of the host cell. This pore-forming property has been identified as a major mechanism by which protein toxins can damage cells. The name alpha-hemolysin derives from early observations that established lytic activity of the toxin on red blood cells. It is expected now that, if applied in sufficient dosage, alpha-hemolysin can permeate any mammalian cell membrane. Although for most of the human population secretion of alpha-hemolysin does not pose a serious health risk, severe staphylococcal infection can cause hemostasis disturbances, thrombocytopenia, and pulmonary lesions (). The crystallographic structure () of the assembled alpha-hemolysin revealed a heptametric organization of the channel. The protein has a mushroom-like shape, with a 50A beta-barrel stem protruding from the cap domain through the lipid bilayer into the cell's interior. The cap of the protein conceals a large vestibule connected to the cell's exterior through a large opening at the top of the cap. The narrowest (1.4~nm in diameter) part of the channel is located at the base of the stem, where the beta-barrel pore connects to the vestibule. Seven side channels lead from the vestibule to the cell's exterior, exiting near the membrane surface. The figure illustrates the 268,000-atom model of alpha-hemolysin in its native environment - a lipid bilayer.
- Click here for a movie (mpeg, 4.0M) showing alpha-hemolysin assembled with a lipid bilayer.
Biotechnological Applications of Alpha-Hemolysin
Several properties of alpha-hemolysin make this membrane channel suitable for various biotechnological applications: assembled alpha-hemolysin is stable over a wide range of pH and temperature, its transmembrane pore stays open at normal conditions, alpha-hemolysin can bind to various biological or synthetic lipid bilayers, the binding proceeds spontaneously and does not require specific ionic conditions.
Delivery Systems. The transmembrane pore of alpha-hemolysin can facilitate controlled delivery of ions and small organic compounds such as sugars or nucleotides across a cell's plasma membrane or through the walls of synthetic lipid vesicles. Using genetically engineered alpha-hemolysins, for which assembly and conductance can be triggered or switched on or off by external biochemical or physical stimuli including light, a lipid bilayer can be made permeable for small solutes at will (, ).
Stochastic Sensors. Suspended in a lipid bilayer, an alpha-hemolysin channel becomes a stochastic sensor when a molecular adapter is placed inside its genetically re-engineered stem, influencing the transmembrane ionic current induced by an applied voltage bias. Reversible binding of analytes to the molecular adapter transiently reduces the ionic current. The magnitude of the current reduction indicates the type of analyte, while the frequency of the current reduction intervals reflects analyte concentration. Such stochastic sensors were demonstrated to simultaneously measure, with a single sensor element, concentrations of several organic analytes () and solution concentrations of two or more divalent metal ions (). The nanometer size pore of alpha-hemolysin was used in another type of stochastic sensor to simultaneously determine concentrations of two different proteins ().
DNA sequencing. The transmembrane pore of alpha-hemolysin can conduct not only small solutes, but also rather big (tens of kDa) linear macromolecules. Thus, driven by a transmembrane potential, DNA or RNA strands can translocate through the pore of alpha-hemolysin, producing the ionic current blockades that reflect the chemical structure of individual strands (). Statistical analysis of many such blockage currents allowed the researchers to discriminate different sequences of RNA () and DNA () homopolymers, as well as the segments of purine and pyrimidine nucleotides within a single RNA molecule (). A single nucleotide resolution has been demonstrated for DNA hairpins (, ), raising the prospect of creating a nanopore sensor capable of reading the nucleotide sequence directly from a DNA or RNA strand.
Imaging Alpha-Hemolysin with Molecular Dynamics
In a biological cell, membrane channels act like miniature valves regulating the flow of ions and other solutes between intracellular compartments and across the cell's boundary. Assembled in complex circuits, they generate, transmit, and amplify signals orchestrating cell function. To investigate how membrane channels work, life scientists, using an extremely fine pipette, isolate a tiny patch of a cell membrane and, in so-called patch clamp measurements, determine electric currents in response to applied electric potentials. Dramatic increase in computational power and its efficient utilization by NAMD allows one today to reproduce such studies computationally, calculating the permeability of a membrane channel to ions and water directly from its atomic structure. In what is one of the largest molecular dynamics simulation to date, described in a recent paper as well as on our web site (here), one copy of the membrane channel alpha-hemolysin, submerged in a lipid membrane and water, was subjected to an external electric field that drove ions and water through the channel. The calculations produced also an image of the electrostatic potential across the channel (see figure).
Imaging alpha-hemolysin with molecular dynamics: Ionic conductance, osmotic permeability and the electrostatic potential map. Aleksij Aksimentiev and Klaus Schulten. Biophysical Journal, 88:3745-3761, 2005.
Orientation discrimination of single stranded DNA inside the a-hemolysin membrane channel. Jerome Mathé, Aleksei Aksimentiev, David R. Nelson, Klaus Schulten, and Amit Meller. Proceedings of the National Academy of Sciences, USA, 102:12377-12382, 2005.
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