MspA
| MspA | |
|---|---|
| Identifiers | |
| Organism | Mycolicibacterium smegmatis (formerly Mycobacterium smegmatis) |
| Symbol | mspA (UniProt A0QR29) |
| PDB structure | 1UUN |
| Properties | |
| Pore diameter | About 1.2 nm at the constriction, which is no more than 0.6 nm long |
| Subunits | 8 (homo-octamer; 184 residues each after the signal peptide) |
| Function | Outer-membrane porin for hydrophilic nutrients; single-molecule sensor in nanopore research |
MspA (Mycobacterium smegmatis porin A) is a protein channel that lets nutrients through the outer membrane of the soil bacterium Mycolicibacterium smegmatis. Eight identical subunits form a goblet whose constriction is about 1.2 nm across and no more than 0.6 nm deep. A mutant of the channel registered single strands of DNA in 2008, in the laboratory of Jens Gundlach at the University of Washington, and in 2012 the same laboratory read known sequences base by base with an enzyme pulling the strand through. The constriction is so short that the current depends on only about four bases at a time, fewer than in alpha-hemolysin.[1] The pore of the best-known commercial sequencer is a different one, CsgG.
Discovery
Michael Niederweis and colleagues purified a porin from M. smegmatis in 1999 and cloned its gene, mspA. The gene encodes a mature protein of 184 amino acids, whose monomer has a mass of 19,406 Da, and the monomers assemble into an oligomer so stable that it survives boiling in 80% dimethyl sulfoxide for 15 minutes before it falls apart.[2] The authors saw no resemblance to any known protein and called MspA "the prototype of a new class of channel-forming proteins". In 2001 the same group reported that MspA provides the main hydrophilic pathway through the cell wall of the bacterium.[3]
The epithet of the species is the genitive of the Latin smegma, a cleansing ointment and, in biology, the sebaceous secretion; Trevisan published the name Bacillus smegmatis in 1889.[4] Since a later reclassification of the mycobacteria the genus has been Mycolicibacterium, and the protein kept its name.
Structure
The crystal structure, solved in 2004 and deposited as 1UUN, showed "a homooctameric goblet-like conformation with a single central channel". It was the first structure of a mycobacterial outer-membrane protein, and none related to it was in the Protein Data Bank. Two consecutive β-barrels with nonpolar outer surfaces form a ribbon around the protein, which the paper called "too narrow to fit the thickness of the mycobacterial outer membrane in contemporary models".[5]
The wide end of the goblet sits on the outer face of the membrane. Its narrowest part, the constriction, is formed by the side chains of two residues from each subunit and measures about 1.2 nm across by no more than 0.6 nm in length, about an eighth of the length of the 5 nm stem of alpha-hemolysin; other papers give about 1 nm by 0.5 nm.[6][7] Fewer bases lie in a short constriction at once. Where about 12 bases sit in the stem of alpha-hemolysin, the current through MspA depends on about four.[6][8]
Engineering
Wild-type MspA is a poor sensor of DNA, because the aspartates lining its constriction repel the negatively charged strand. Butler and colleagues replaced three of them with asparagine (D90N, D91N and D93N), removing the negative charge from the constriction, and the mutant took up and registered single-stranded DNA. A second mutant, which also swapped negatively charged residues in the vestibule for positive ones, interacted with DNA about 20 times as often, needed half the voltage and held the strand in the vestibule about 100 times longer.[1]
An octamer repeats every mutation eight times. In 2012 a group joined two monomers with a peptide linker of 17 to 62 amino acids and showed that the dimers still assembled into working channels with the same blockades for DNA hairpins, a first step toward pores whose subunits differ.[7]
Use in sequencing
| Year | Result |
|---|---|
| 2008 | Single-stranded DNA detected with an engineered MspA[1] |
| 2010 | All four nucleotides distinguished, and single nucleotides resolved when double-stranded DNA held them in the constriction[9] |
| 2011 | DNA held in the pore by a biotin-NeutrAvidin complex gave much larger current differences between homopolymers of A, C, T and G than in alpha-hemolysin, and methylated cytosine was distinguishable from unmethylated[10] |
| 2012 | Phi29 DNA polymerase pulling DNA through MspA gave current levels lasting a median of 28 ms and differing by up to 40 pA, in readable regions of 42 to 53 nucleotides[11] |
| 2013 | 5-methylcytosine and 5-hydroxymethylcytosine mapped in single strands, with a single-read detection efficiency of 97.5% and 97%[12] |
| 2014 | A map of the current for all 256 four-nucleotide combinations; reads of phage phi X 174 up to 4,500 bases that matched the reference genome[8] |
| 2015 | Steps of the helicase Hel308 followed along DNA with about 40 pm sensitivity, against about 300 pm for FRET and optical tweezers[13] |
| 2021 | A DNA-peptide conjugate pulled through MspA by Hel308 and read again and again, with single amino acid substitutions told apart[14] |
The 2015 entry in the table used the pore as a ruler. Because the current through MspA changes with each step of a bound enzyme, the authors could follow the helicase Hel308 along a strand with about 40 pm sensitivity, and they proposed the method for any protein that translocates on DNA or RNA, including helicases, polymerases, recombinases and repair enzymes.[13]
The peptide work of 2021 turned the pore toward proteins. Hel308 pulls the strand in steps of about 0.33 nm, and the authors rewound a single peptide until it had been read 117 times. With more than about 30 rereads of one molecule, the error rate in identifying a single amino acid variant fell below one in a million.[14] The motor proteins that make such reading possible have their own article.
Limits
MspA needs a membrane, and the usual one is a lipid bilayer. Oxford Nanopore Technologies announced in 2016 that its R9 flow cells used CsgG, a curli secretion channel with a 0.9 nm constriction, licensed from a laboratory at the VIB institute in Belgium and from University College London.[15]
At Molecular Reality
Molecular Reality Corporation works with solid-state pores and calls protein pores "delicate flowers".[16] Its atlas of molecular-sensing groups, The Hole World, draws each of its 2,112 entries (in the build of September 26, 2026) with one of 15 pore icons. Of these, 811 are silicon nitride, 33 are alpha-hemolysin and two are CsgG. One entry is drawn as MspA, the University of Washington group in Seattle that turned the pore into a sequencing tool.[17]
See also
References
- ^ a b c Butler, T. Z.; Pavlenok, M.; Derrington, I. M.; Niederweis, M.; Gundlach, J. H. (2008). "Single-molecule DNA detection with an engineered MspA protein nanopore". Proceedings of the National Academy of Sciences 105 (52): 20647-20652. doi:10.1073/pnas.0807514106.
- ^ Niederweis, M.; et al. (1999). "Cloning of the mspA gene encoding a porin from Mycobacterium smegmatis". Molecular Microbiology 33 (5): 933-945. doi:10.1046/j.1365-2958.1999.01472.x.
- ^ Stahl, C.; et al. (2001). "MspA provides the main hydrophilic pathway through the cell wall of Mycobacterium smegmatis". Molecular Microbiology 40 (2): 451-464. doi:10.1046/j.1365-2958.2001.02394.x.
- ^ "Species Mycolicibacterium smegmatis". List of Prokaryotic names with Standing in Nomenclature. DSMZ. Retrieved October 25, 2026.
- ^ Faller, M.; Niederweis, M.; Schulz, G. E. (2004). "The structure of a mycobacterial outer-membrane channel". Science 303 (5661): 1189-1192. doi:10.1126/science.1094114.
- ^ a b Deamer, D.; Akeson, M.; Branton, D. (2016). "Three decades of nanopore sequencing". Nature Biotechnology 34 (5): 518-524. doi:10.1038/nbt.3423.
- ^ a b Pavlenok, M.; et al. (2012). "MspA nanopores from subunit dimers". PLoS ONE 7 (6): e38726. doi:10.1371/journal.pone.0038726.
- ^ a b Laszlo, A. H.; et al. (2014). "Decoding long nanopore sequencing reads of natural DNA". Nature Biotechnology 32 (8): 829-833. doi:10.1038/nbt.2950.
- ^ Derrington, I. M.; et al. (2010). "Nanopore DNA sequencing with MspA". Proceedings of the National Academy of Sciences 107 (37): 16060-16065. doi:10.1073/pnas.1001831107.
- ^ Manrao, E. A.; et al. (2011). "Nucleotide discrimination with DNA immobilized in the MspA nanopore". PLoS ONE 6 (10): e25723. doi:10.1371/journal.pone.0025723.
- ^ Manrao, E. A.; et al. (2012). "Reading DNA at single-nucleotide resolution with a mutant MspA nanopore and phi29 DNA polymerase". Nature Biotechnology 30 (4): 349-353. doi:10.1038/nbt.2171.
- ^ Laszlo, A. H.; et al. (2013). "Detection and mapping of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore MspA". Proceedings of the National Academy of Sciences 110 (47): 18904-18909. doi:10.1073/pnas.1310240110.
- ^ a b Derrington, I. M.; et al. (2015). "Subangstrom single-molecule measurements of motor proteins using a nanopore". Nature Biotechnology 33 (10): 1073-1075. doi:10.1038/nbt.3357.
- ^ a b Brinkerhoff, H.; Kang, A. S. W.; Liu, J.; Aksimentiev, A.; Dekker, C. (2021). "Multiple rereads of single proteins at single-amino acid resolution using nanopores". Science 374 (6574): 1509-1513. doi:10.1126/science.abl4381.
- ^ "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.
- ^ "The Hole World". Molecular Reality Corporation. September 2026.
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