Ultrasensitive detection of protein translocated through toxin pores in droplet-interface bilayers

Abstract

Many bacterial toxins form proteinaceous pores that facilitate the translocation of soluble effector proteins across cellular membranes. With anthrax toxin this process may be monitored in real time by electrophysiology, where fluctuations in ionic current through these pores inserted in model membranes are used to infer the translocation of individual protein molecules. However, detecting the minute quantities of translocated proteins has been a challenge. Here, we describe use of the droplet-interface bilayer system to follow the movement of proteins across a model membrane separating two submicroliter aqueous droplets. We report the capture and subsequent direct detection of as few as 100 protein molecules that have translocated through anthrax toxin pores. The droplet-interface bilayer system offers new avenues of approach to the study of protein translocation.

Ratcheting up protein translocation with anthrax toxin

“…First, its three protein components can be expressed recombinantly and studied independently. Second, the protein translocase and the individual steps of substrate translocation can be readily reconstituted from purified proteins and studied using planar bilayer electrophysiology at the ensemble21, 22, 38, 49, 50‐58 and single‐molecule level 22, 40, 49, 51, 59. Critical to this electrophysiological approach (also used in other systems60‐67) is the ability to precisely control the driving force and solution conditions on either side of the membrane.…”

“…Third, for a membrane‐protein system, structural studies using X‐ray crystallography are tractable, because the translocase also exists in a soluble state 38, 40, 42, 68. In this manner, researchers have been able to obtain structural information35, 36, 38‐40, 42, 68‐70 and distinguish possible translocation models using a wide variety of functional assays 21, 22, 38, 49‐58, 71…”

Anthrax toxin-induced rupture of artificial lipid bilayer membranes

“…39,43,50,53 Experimentally proving translocation of a trace amount of protein is more difficult, because there is no means to amplify protein. Fischer and colleagues claimed that they could detect as few as 100 LF N proteins (800 aM) at a rate of ≈2 s −1 with the channel, 80 while the mean time to detect individual LF N molecules at that low concentration is ≈10 6 s. 81 LF N translocation through the PA 63 channel 28, 30-32, 80, 82-87 has been hypothesized to occur either by electrodiffusion as charged rods 75 or via a "Brownian ratchet." 20 Both models make assumptions about how the energy barriers to LF N transport are overcome.…”

Bordetella pertussis adenylate cyclase toxin translocation across a tethered lipid bilayer

“…These latter toxins provide a unique opportunity to analyze the molecular mechanisms and the physicochemical principles underlying polypeptide transport across biological membranes. Studies combining structural, biochemical, and electrophysiological approaches have begun to unravel the various strategies developed by these toxins to deliver their catalytic moieties across the cell membranes (5)(6)(7)(8)(9)(10).…”