Alpha-hemolysin
| Alpha-hemolysin | |
|---|---|
| Identifiers | |
| Organism | Staphylococcus aureus |
| Symbol | hla (UniProt P09616) |
| PDB structure | 7AHL |
| Properties | |
| Pore diameter | 1.4 nm at the narrowest point, 4.6 nm at the widest |
| Subunits | 7 (homoheptamer; 293 residues and 33.3 kDa each) |
| Function | Pore-forming toxin; single-molecule sensor in nanopore research |
Alpha-hemolysin (α-hemolysin, α-toxin, Hla) is a protein secreted by the bacterium Staphylococcus aureus. Seven copies assemble in a cell membrane into a mushroom-shaped channel about 10 nm long that lets small molecules leak out of the cell and kills it. In 1996 the same channel, set in a lipid bilayer, became the first biological nanopore to register single strands of DNA and RNA passing through it, and it was the pore of much of the early work on nanopore sequencing.[1]
The channel is 1.4 nm wide at its narrowest point. A single strand of DNA or RNA fits through it and double-stranded DNA does not, a fact that decided what the first experiments could measure. Commercial sequencers have since moved to other pores, but alpha-hemolysin is still used where a pore of known structure that can be changed one residue at a time is wanted.
Toxin
The gene is hla (UniProt lists hly first). Hla is made as a precursor of 319 amino acids; a signal peptide of 26 residues is cut off, and the mature chain has 293 residues and a mass of 33.3 kDa.[2][3] Monomers are soluble. They bind the membrane of a target cell, assemble first into an oligomer that cannot yet lyse it, and then into the heptamer that punches through the bilayer.[2] On human cells the toxin binds the metalloprotease ADAM10, and the binding is required for pore formation, for the loss of focal adhesions and for cell damage.[4]
The name comes from red blood cells, which the toxin lyses. Rabbit red cells bind much more of it than human ones do.[2]
Structure
The crystal structure of the heptamer, solved to 1.9 Å in 1996, shows a mushroom. A cap of seven β-sandwiches sits outside the membrane, a rim lies against the bilayer, and a stem crosses it. The stem is the lower half of a barrel of 14 antiparallel β-strands, to which each subunit contributes two strands 6.5 nm long. The inside of the barrel is mostly hydrophilic and its outside carries a hydrophobic belt 2.8 nm wide, which sits in the lipid.[5] A solvent-filled channel runs 10 nm along the sevenfold axis and varies from 1.4 nm to 4.6 nm in diameter, widest inside the cap and narrowest at the central constriction, which later work used as a recognition site for single bases.[5][6] Its coordinates are entry 7AHL in the Protein Data Bank.[3]
The DNA paper appeared on November 26, 1996, and described the channel as 2.6 nm in diameter. Seventeen days later the structure was published.[1][5]
Use as a sensor
In the 1996 experiment, with 1 M potassium chloride on both sides of the bilayer and −120 mV across it, the open channel carried a current of 120 pA. A molecule of poly(U) with a mean length of 210 bases cut the current by 85 to 100% for several hundred to several thousand microseconds, and the time was proportional to the length of the polymer.[1] David Deamer's account has him visit Kasianowicz at NIST in late 1993 to test whether RNA homopolymers could be driven through a single channel. Blockades appeared as soon as the polymers were added on the cis side and a positive bias above 80 mV was applied to the trans side. Preliminary diffraction results from the group solving the structure reached him and Branton shortly afterward, and put the channel between about 1.5 and 2.5 nm wide, which fit single strands passing and double-stranded DNA staying out.[7] A Harvard patent application filed on March 17, 1995 names George Church, Deamer, Daniel Branton, Richard Baldarelli and John Kasianowicz as inventors and lists alpha-hemolysin and maltoporin as preferred channels.[8] Church is also an investor in Molecular Reality Corporation.[9]
| Year | Result | Source |
|---|---|---|
| 1996 | Single strands of RNA and DNA driven through one channel; blockade time proportional to length | [1] |
| 1999 | Pyrimidine and purine segments of one RNA molecule told apart on a microsecond time scale | [10] |
| 2001 | DNA hairpins that differ by one base pair, or by one nucleotide, discriminated | [11] |
| 2003 | Double-stranded DNA unzipped by pulling one strand through the pore | [12] |
| 2009 | All four DNA bases identified at three recognition sites in the barrel, in a strand held in place | [13] |
| 2009 | Unlabelled nucleoside monophosphates identified one at a time with an average accuracy of 99.8% | [14] |
| 2012 | DNA ratcheted through the pore by a polymerase at median rates of 2.5 to 40 nucleotides per second | [15] |
Because the channel is a homoheptamer, a point mutation in the gene appears seven times in the pore, and laboratories exploited this to change the lumen. Gu and colleagues in Hagan Bayley's laboratory lodged a cyclodextrin in the channel as a molecular adapter and read single binding events from the fluctuations of the current, an approach known as stochastic sensing.[16] The unzipping experiment of 2003 relies on the 1.4 nm constriction: the second strand cannot enter, so it is stripped off as the first is pulled through.[12]
Limits
The diameter is fixed by the sequence of the protein, and the pore sits in a lipid bilayer, which is harder to make and keep than the pore. Oxford Nanopore Technologies announced its R9 flow cell in 2016 with a different pore, CsgG, and a sequencing speed of about 250 bases per second per pore.[17] The guide published by Molecular Reality Corporation, which builds solid-state nanopores, puts the trade in one line: "Protein pores are delicate flowers."[18]
A March 2026 post by the founder, on DNA labeled at selected bases with osmium tetroxide 2,2'-bipyridine, recalls that such a strand "could translocate through alpha-hemolysin. The constriction site looks too narrow for this to happen, but it does happen."[19]
See also
References
- ^ a b c d Kasianowicz, J. J.; Brandin, E.; Branton, D.; Deamer, D. W. (1996). "Characterization of individual polynucleotide molecules using a membrane channel". Proceedings of the National Academy of Sciences 93 (24): 13770-13773. doi:10.1073/pnas.93.24.13770. PMID 8943010.
- ^ a b c "P09616: Alpha-hemolysin, Staphylococcus aureus". UniProt. Retrieved October 25, 2026.
- ^ a b "7AHL: Alpha-hemolysin from Staphylococcus aureus, heptameric pore". RCSB Protein Data Bank. Retrieved October 25, 2026.
- ^ Wilke, G. A.; Bubeck Wardenburg, J. (2010). "Role of a disintegrin and metalloprotease 10 in Staphylococcus aureus α-hemolysin-mediated cellular injury". Proceedings of the National Academy of Sciences 107 (30): 13473-13478. doi:10.1073/pnas.1001815107.
- ^ a b c Song, L.; Hobaugh, M. R.; Shustak, C.; Cheley, S.; Bayley, H.; Gouaux, J. E. (1996). "Structure of staphylococcal α-hemolysin, a heptameric transmembrane pore". Science 274 (5294): 1859-1865. doi:10.1126/science.274.5294.1859. PMID 8943190.
- ^ Stoddart, D.; et al. (2010). "Nucleobase recognition in ssDNA at the central constriction of the α-hemolysin pore". Nano Letters 10 (9): 3633-3637. doi:10.1021/nl101955a.
- ^ Deamer, D.; Akeson, M.; Branton, D. (2016). "Three decades of nanopore sequencing". Nature Biotechnology 34 (5): 518-524. doi:10.1038/nbt.3423. PMID 27153285.
- ^ "US5795782A: Characterization of individual polymer molecules based on monomer-interface interactions". Google Patents. August 18, 1998. Retrieved October 25, 2026.
- ^ "Company records". Molecular Reality Corporation. October 2026.
- ^ Akeson, M.; et al. (1999). "Microsecond time-scale discrimination among polycytidylic acid, polyadenylic acid, and polyuridylic acid as homopolymers or as segments within single RNA molecules". Biophysical Journal 77 (6): 3227-3233. doi:10.1016/S0006-3495(99)77153-5. PMID 10585944.
- ^ Vercoutere, W.; et al. (2001). "Rapid discrimination among individual DNA hairpin molecules at single-nucleotide resolution using an ion channel". Nature Biotechnology 19 (3): 248-252. doi:10.1038/85696.
- ^ a b Sauer-Budge, A. F.; et al. (2003). "Unzipping kinetics of double-stranded DNA in a nanopore". Physical Review Letters 90 (23): 238101. doi:10.1103/PhysRevLett.90.238101.
- ^ Stoddart, D.; et al. (2009). "Single-nucleotide discrimination in immobilized DNA oligonucleotides with a biological nanopore". Proceedings of the National Academy of Sciences 106 (19): 7702-7707. doi:10.1073/pnas.0901054106.
- ^ Clarke, J.; et al. (2009). "Continuous base identification for single-molecule nanopore DNA sequencing". Nature Nanotechnology 4 (4): 265-270. doi:10.1038/nnano.2009.12.
- ^ Cherf, G. M.; et al. (2012). "Automated forward and reverse ratcheting of DNA in a nanopore at 5-Å precision". Nature Biotechnology 30 (4): 344-348. doi:10.1038/nbt.2147.
- ^ Gu, L. Q.; et al. (1999). "Stochastic sensing of organic analytes by a pore-forming protein containing a molecular adapter". Nature 398 (6729): 686-690. doi:10.1038/19491.
- ^ "Update: New "R9" nanopore for faster, more accurate sequencing, and new ten minute preparation kit". Oxford Nanopore Technologies. May 26, 2016. Retrieved October 25, 2026.
- ^ "Keltar's Field Guide to Molecular Reality". Molecular Reality Corporation. August 2026.
- ^ Kemmishtree (March 29, 2026). "Magic with labels". EQBio-OS.
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