UC Berkeley

Transmembrane protein translocation is a fundamental and ubiquitous process that operates under several conserved principles. Protein conducting machines, known as translocase channels, catalyze the unfolding and subsequent transport of substrate proteins through narrow pores in an energy-dependent manner. Investigating how translocase machines rectify external energy inputs is key to understanding their force transduction mechanisms and overall function. Molecular ratchets are believed to be an integral mechanism for providing directional polypeptide transport through translocase channels, and several types of ratchets have been identified in these machines.

In this work, anthrax toxin, a protein translocase that possesses a hydrophobic gasket known as the φ clamp, is used as a model system to investigate the role of hydrophobic/steric ratcheting in

protein translocation. Planar lipid bilayer electrophysiology was used to establish that the PA φ clamp binds hydrophobic/aromatic residues in peptide substrates, and to determine the consequences this binding interaction has on transport of these peptides. Under conditions where protein unfolding is shown to be rate-limiting, sequences identified as φ-clamp interacting increase translocation of folded downstream domains.

Single channel electrophysiology was used to record substrate and driving force dependent changes in the conductance of a single PA channel. Open, bound, and two intermediate conductance states are identified, which we hypothesize correspond to different conformations of the catalytic φ-clamp loop. Crystallographic evidence, which supports this theory, is presented. A kinetic analysis of single channel peptide binding and translocation events shows that the translocation pathway for hydrophobic/aromatic sequences has committed steps. A hydrophobic ratchet model is proposed to explain φ-clamp catalysis and how the favorable interaction between the clamp and hydrophobic/aromatic sequences may favor unfolding and increase directional movement of the translocating chain.