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Technology Topics Cloning

Protein nanopores

PSI-SGKB [doi:10.1038/th_psisgkb.2009.6]
Technical Highlight - March 2009
Short description: Ultra high-speed DNA sequencing could be a step nearer as the protein channel MpsA is found to be an excellent nanopore.Proc. Natl. Acad. Sci. USA 105, 20647-20652 (2008)

Membranes with nanometer-scale holes known as nanopores have great potential as a tool for low-cost ultra high-speed sequencing of nucleic acids that dispenses with the need for polymerases and, in theory, would be unconstrained by DNA length.

Nanopores allow ionic current to flow across the membrane, but this flow is blocked when the channel is entirely occupied by a molecule. Partial blockage, for example, by the different nucleotides in a nucleic acid strand, reduces ionic current by different amounts, and so it should in principle be possible to devise a way of deducing the sequence of a DNA or RNA strand threaded through the pore. In a more general application, pores should be able to record the concentration, structure and dynamics of molecules passed through them.

Although progress has been made using synthetic pores, protein channels are attractive because they self-assemble, which ensures reproducibility from one pore to the next, and the protein can be engineered to suit the analysis.

Experiments using a-hemolysin as the protein channel showed that individual DNA bases could be recognized, but DNA sequencing remained out of reach. So other channel proteins with nanopore potential are being sought.

Butler et al. 1 reveal one promising candidate in the form of the porin protein MspA from Mycobacterium smegmatis. This protein is very stable and its structure is known, which gives a reference point for modifying it in required ways.

For a nanopore to be useful, its diameter must not be much larger than the target molecule. MspA fits the bill as it has a narrow part of the channel about 1 nanometer long and 1 nanometer wide, with a wider opening at each end. It is also amenable to genetic engineering, as a wide range of mutant forms retain high expression levels and channel-forming ability.

The authors designed and constructed two MspA mutants in which negative charge in the constricted region was eliminated, and showed that these proteins can electronically detect and characterize individual molecules of single-stranded DNA as they are electrophoretically driven through the pore.

Identification of this new nanopore for nucleic acid analysis should pave the way for further studies optimizing the interactions between MspA and DNA and provide an important step towards nanopore DNA sequencing and other nanosensor applications.

Maria Hodges


  1. B. Z. Butler, M. Pavlenok, I. M. Derrington, M. Niederweis & J. Gundlach Single-molecule DNA detection with an engineered MspA protein nanopore.
    Proc. Natl. Acad. Sci. USA 105, 20647-20652 (2008).

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