Protein export is an essential mechanism in living cells and exported proteins are usually translocated through a protein-conducting channel in an unfolded state. Here we analyze, by electrical detection, the entry and transport of unfolded proteins, at the single molecule level, with different stabilities through an aerolysin pore, as a function of the applied voltage and protein concentration. The frequency of ionic current blockades varies exponentially as a function of the applied voltage and linearly as a function of protein concentration. The transport time of unfolded proteins decreases exponentially when the applied voltage increases. We prove that the ionic current blockade duration of a double-sized protein is longer than that assessed for a single protein supporting the transport phenomenon. Our results fit with the theory of confined polyelectrolyte and with some experimental results about DNA or synthetic polyelectrolyte translocation through protein channels as a function of applied voltage. We discuss the potential of the aerolysin nanopore as a tool for protein folding studies as it has already been done for α-hemolysin.

The aim of this work is to study pore protein denaturation inside a lipid bilayer and to probe current asymmetry as a function of the channel conformation. We describe the urea denaturation of a-hemolysin channel and the channel formation of a-hemolysin monomer incubated with urea prior to insertion into a lipid bilayer. Analysis of single-channel recordings of current traces reveals a sigmoid curve of current intensity as a function of urea concentration. The normalized current asymmetry at 29 ± 4% is observed between 0 and 3.56 M concentrations and vanishes abruptly down to 0 concentration exceeds 4 M. The loss of current asymmetry through a-hemolysin is due to the denaturation of the channel's cap. We also show that the a-hemolysin pore inserted into a lipid bilayer is much more resistant to urea denaturation than the a-hemolysin monomer in solution: The pore remains in the lipid bilayer up to 7.2 M urea. The pore formation is possible up to 4.66 M urea when protein monomers were previously incubated in urea.

We use single cell force spectroscopy to compare elasticity, adhesion and tether extrusion on four breast cancer cell lines with an increasing invasive potential. We perform cell attachment/detachment experiments either on fibronectin or on another cell using an Atomic Force Microscope. Our study on the membrane tether formation from cancer cells show that they are easier to extrude from aggressive invasive cells. Measured elastic modulus values confirm that more invasive cells are softer. Moreover, the adhesion force increases with the invasive potential. Our results provide a mechanical signature of breast cancer cells that correlates with their invasivity.