title: Nucleobase Recognition by Truncated α-Hemolysin Pores.
abstract: The α-hemolysin (αHL) protein nanopore has been investigated previously as a base detector for the strand sequencing of DNA and RNA. Recent findings have suggested that shorter pores might provide improved base discrimination. New work has also shown that truncated-barrel mutants (TBM) of αHL form functional pores in lipid bilayers. Therefore, we tested TBM pores for the ability to recognize bases in DNA strands immobilized within them. In the case of TBMΔ6, in which the barrel is shortened by ∼16 Å, one of the three recognition sites found in the wild-type pore, R1, was almost eliminated. With further mutagenesis (Met113 → Gly), R1 was completely removed, demonstrating that TBM pores can mediate sharpened recognition. Remarkably, a second mutant of TBMΔ6 (Met113 → Phe) was able to bind the positively charged β-cyclodextrin, am7βCD, unusually tightly, permitting the continuous recognition of individual nucleoside monophosphates, which would be required for exonuclease sequencing mediated by nanopore base identification.
authors.affiliation: Department of Chemistry, University of Oxford , Oxford, OX1 3TA, United Kingdom.
authors.forename: Mariam
authors.initials: M
authors.lastname: Ayub
authors.affiliation: Department of Chemistry, University of Oxford , Oxford, OX1 3TA, United Kingdom.
authors.forename: David
authors.initials: D
authors.lastname: Stoddart
authors.affiliation: Department of Chemistry, University of Oxford , Oxford, OX1 3TA, United Kingdom.
authors.forename: Hagan
authors.initials: H
authors.lastname: Bayley
mesh_terms: D000225:Adenine / Q000032:analysis* / Q000737:chemistry; D019943:Amino Acid Substitution; D001483:Base Sequence; D015374:Biosensing Techniques; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics; D008051:Lipid Bilayers / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020102:Mycobacterium smegmatis / Q000737:chemistry; D058608:Nanopores / Q000648:ultrastructure; D017354:Point Mutation; D011066:Poly C / Q000032:analysis* / Q000737:chemistry; D018272:Porins / Q000737:chemistry* / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D055672:Static Electricity; D047392:beta-Cyclodextrins / Q000737:chemistry
chemical_list: D006460:Hemolysin Proteins; D008051:Lipid Bilayers; D018272:Porins; D011994:Recombinant Proteins; D047392:beta-Cyclodextrins; C121838:mspA protein, Mycobacterium smegmatis; C023826:poly(dC); D011066:Poly C; D000225:Adenine; C031215:betadex
keywords: alpha-hemolysin; base identification; nanopore; toroidal lipid pore; truncated pore