Clostridium botulinum outbreaks during the “Golden Age” of European science led to the discovery of these microbial neurotoxins, which are the most toxic substance to man. Wartime efforts identified the protein toxin that was later found to enter nerves and short circuit their electrochemical signals. As a 2-chain AB toxin, the Botulinum neurotoxin heavy chain translocates the enzymatic light chain across membranes. Today, rebranded as Botox, the Botulinum neurotoxins are in wide use for medical and cosmetic applications. While their physiological effects are well understood, their mechanism of cellular entry remains poorly understood owing to the difficulty capturing and observing transport intermediates. To enable mechanistic studies of translocation, we have developed a FRET biosensor that measures the dissipation of transmembrane gradients with single liposome resolution, which facilitates identifying active proteins within the ensemble. Here, we leverage this system to dissect the underlying translocation mechanism of Botox. We show that efficiency of pore formation depends on proteolytic processing of the holotoxin, and is enhanced by the presence of known co-receptors. Site-specific labeling of the toxin allowed us to count the number of proteins associated with pore formation to reveal the stoichiometry of the pore for the first time. The current pharmacological agent is a natural product with variable results while there still is no treatment for botulism paralysis. We aim to harness our single molecule approaches to help develop next generation treatments and characterize potential therapeutics.
zhang et al. (Sun,) studied this question.