Three reversible and controllable discrete steps of channel gating of a viral DNA packaging motor

The channel of the viral DNA packaging motor allows dsDNA to enter the protein procapsid shell during maturation and to exit during infection. We recently showed that the bacteriophage phi29 DNA packaging motor exercises a one-way traffic property using a channel as a valve for dsDNA translocation. This raises a question of how dsDNA is ejected during infection if the channel only allows the dsDNA to travel inward. We proposed that DNA forward or reverse travel is controlled by conformational changes of the channel. Here we reported our direct observation that the channel indeed exercises conformational changes by single channel recording at a single-molecule level. The changes were induced by high electrical voltage, or by affinity binding to the C-terminal wider end located within the capsid. Novel enough, the conformational change of the purified connector channel exhibited three discrete gating steps, with a size reduction of 32% for each step. We investigated the role of the terminal and internal loop of the channel in gating by different mutants. The step-wise conformational change of the channel was also reversible and controllable, making it an ideal nano-valve for constructing a nanomachine with potential applications in nanobiotechnology and nanomedicine.