Anthrax toxin is comprised of three proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF). PA oligomerizes, inserts into the membrane, and forms a proton motive force (PMF)-driven channel that translocates LF and EF into host cells. The channel contains nonspecific polypeptide clamps, which catalyze unfolding and translocation. How the collective action of these clamps transduces the PMF into a productive mechanical force while avoiding tightly bound kinetic traps is unclear. A Brownian-ratchet model posits that the translocating chain is consistently in the “extended-chain” configuration during force transduction. The “helix-compaction” model proposes force is transduced as part of the chain transiently compresses into a helical conformation. Clamp allostery and substrate helix-to-coil/coil-to-helix cooperativity are postulated to facilitate the major conformational transitions in the latter model. Here ~40-60-residue LF peptides were synthesized with identical sequences but variable stereochemistries to control for side-chain chemistry while manipulating helical sense and helix-to-coil/coil-to-helix cooperativity. Peptide binding, translocation, and dissociation were measured using single-channel electrophysiology. While isotactic peptides with relatively pure stereochemistry translocated rapidly on the sub-sec timescale through a series of consecutive irreversible steps, syndiotactic peptides containing alternating ʟ- and ᴅ-enantiomer sequences translocated through a less-complex mechanism that was slower than the 776-residue LF. Allosteric binding, observed only with the isotactic ʟ-enantiomer sequence, depended on acidic pH and functional peptide-clamp sites. These results favor a model whereby allosteric clamps engage and release a substrate chain through stereospecific contacts in a manner that supports PMF-dependent unidirectional translocation along the length of the peptide chain.