Journal
ACS NANO
Volume 9, Issue 9, Pages 9050-9061Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acsnano.5b03053
Keywords
protein nanopore; electrical detection autotransporters; secretion; single molecule; translocation; unfolded protein
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Funding
- National Institutes of Health [R01 GM097573, U54 GM105816]
- Notre Dame CBBI Graduate Fellowship [T32 GM075762]
- Agence Nationale de la Recherche [ANR 12-NANO-0012-03]
- Region Ile-de-France in the Framework of DIM Nano-K [094251]
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To evaluate the physical parameters governing translocation of an unfolded protein across a lipid bilayer, we studied protein transport through aerolysin, a passive protein channel, at the single-molecule level. The protein model used was the passenger domain of pertactin, an autotransporter virulence protein. Transport of pertactin through the aerolysin nanopore was detected as transient partial current blockades as the unfolded protein partially occluded the aerolysin channel. We compared the dynamics of entry and transport for unfolded pertactin and a covalent end-to-end dimer of the same protein. For both the monomer and the dimer, the event frequency of current blockades increased exponentially with the applied voltage, while the duration of each event decreased exponentially as a function of the electrical potential. The blockade time was twice as long for the dimer as for the monomer. The calculated activation free energy includes a main enthalpic component that we attribute to electrostatic interactions between pertactin and the aerolysin nanopore (despite the low Debye length), plus an entropic component due to confinement of the unfolded chain within the narrow pore. Comparing our experimental results to previous studies and theory suggests that unfolded proteins cross the membrane by passing through the nanopore in a somewhat compact conformation according to the blob model of Daoud and de Gennes.
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