Dynamics and Energy Contributions for Transport of Pertactin through an Aerolysin Nanopore

Abstract

Autotransporters are a large family of extracellular monomeric virulence proteins from Gram-negative bacteria. Despite their simplicity, many aspects of the autotransporter secretion mechanism remain unclear. We are using pertactin, an archetypical autotransporter from Bordetella pertussis, as a model for secretion studies. The final step of autotransporter secretion is C-to-N-terminal transport of the central passenger domain through the outer membrane, mediated by the C-terminal translocator domain. Passenger folding occurs only after this final secretion step, which requires neither ATP nor a proton gradient. Passenger folding may therefore serve as a driving force for pertactin secretion. For this reason, it is interesting to consider how autotransporters are secreted through their own translocator domain to the cell surface.As a first step, we are mimicking this transport using a simpler model consisting of a well-known nanopore. Transport of the pertactin passenger is detected at the single molecule level using electrophysiological techniques. We show that unfolded pertactin dynamics through a single aerolysin pore can be described using a model developed for an unrelated protein. A Van’t Hoff-Arrhenius law describes the frequency of blockades as a function of the applied voltage. The unfolded chains are dominated by an activation energy that has both an entropic component and an enthalpic origin. We compare our experimental results to theory and show that proteins cross the membrane by passing through the aerolysin nanopore. We have used these results to develop a general description of the comportment of an unfolded protein during its transport through a protein nanopore.