The partitioning of polypeptides into nanoscale transmembrane pores is of fundamental importance in biology. Examples include protein translocation in the endoplasmic reticulum and the passage of proteins through the nuclear pore complex. Here we examine the exchange of cationic α-helical peptides between the bulk aqueous phase and the transmembrane β-barrel of the α-hemolysin (αHL) protein pore at the single-molecule level. The experimental kinetic data suggest a two-barrier, single-well free energy profile for peptide transit through the αHL pore. This free energy profile is strongly voltage- and peptide-length-dependent. We used the Woodhull-Eyring formalism to rationalize the values measured for the association and dissociation rate constants kon and koff and to separate koff into individual rate constants for exit through each of the openings of the protein pore. The rate constants kon, k offcis. and kofftrans decreased with the length of the peptide. At high transmembrane potentials, the alanine-based peptides, which include bulky lysine side chains, bind more strongly (formation constants Kf ∼ tens of M-1) than highly flexible polyethylene glycols (Kf ∼ M-1) to the lumen of the αHL protein pore. In contrast, at zero transmembrane potential, the peptides bind weakly to the lumen of the pore, and the affinity decreases with the peptide length, similar to the case of the polyethylene glycols. The binding is enhanced at increased transmembrane potentials, because the free energy contribution ΔG = -ζδ FV/RT predominates with the peptides. We suggest that the αHL protein may serve as a robust and versatile model for examining the interactions between positively charged signal peptides and a β-barrel pore.
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