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Albany 2013: Book of Abstracts

category image Albany 2013
Conversation 18
June 11-15 2013
©Adenine Press (2012)

Polymers through pores: single-molecule experiments with nucleic acids, polypeptides and polysaccharides

The translocation of polymers through pores has been examined for almost two decades, with an emphasis on nucleic acids. There are also interesting circumstances in biology where polypeptides and polysaccharides pass through transmembrane pores, and our laboratory has been investigating examples of them.

Single-molecule nucleic sequencing by nanopore technology is an emerging approach for ultrarapid genomics. Strand sequencing with engineered protein nanopores is a viable technology, which has required advances in four areas: nucleic acid threading, nucleobase identification, controlled strand translocation, and nanopore arrays (Bayley, 2012). The latter remain a pressing need, and our attempts to improve arrays will be described.

In several physiological situations folded proteins pass through transmembrane pores. We have developed a model system, comprising mutant thioredoxins, as the translocated proteins, and staphylococcal alpha-hemolysin, as the pore. Our findings support a mechanism in which there is local unfolding near the terminus of the polypeptide that enters the pore. The remainder of the protein then unfolds spontaneously and diffuses through the pore into the recipient compartment (Rodriguez-Larrea and Bayley, 2013).

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We have also examined the pore formed by the E. coli outer membrane protein Wza, which transports capsular polysaccharide from its site of synthesis to the outside of the cell. We made mutant open forms of the pore and screened blockers for them by electrical recording in planar bilayers. The most effective blocker binds in the alpha-helix barrel of Wza, a site accessible from the external medium, and therefore a prospective target for antibiotics (Kong et al., 2013).

References

    Bayley, H. (2012). Are we there yet? Phys Life Rev 9, 161-163.

    Kong, L., Harrington, L., Li, Q., Cheley, S., Davis, B.G., and Bayley, H. (2013). Single-molecule interrogation of a bacterial sugar transporter allows the discovery of an extracellular inhibitor. Nature Chemistry, in press.

    Rodriguez-Larrea, D., and Bayley, H. (2013). Multistep protein unfolding during nanopore translocation. Nature Nanotechnology, in press.


Hagan Bayley

Department of Chemistry
University of Oxford Oxford, OX1 3TA, UK

Ph: +44 1865 285101
hagan.bayley@chem.ox.ac.uk