FIG 5.

Pore formation by PFO^N197C at low temperature modified with NEM before and after prepore assembly. (A) Pore formation was monitored over time by CF release from liposomes treated with PFO (±NEM), PFO^N197W, PFO^N197C (±NEM), and DLY at 6°C. CF release by PFO and PFO^N197C was monitored for 1,000 s to allow for prepore formation, and then N-ethylmaleimide (NEM) was injected (final concentration of 100 μM) to modify the sulfhydryl of N197C. As shown, NEM triggers the rapid conversion of prepore PFO^N197C but has no effect on the low rate of pore formation by PFO. (B) The cysteine sulfhydryl of PFO^N197C was labeled with NEM prior to injecting it into liposomes at 6°C compared to PFO. Note that the rate of pore formation by PFO^N197C is higher when the prepore is allowed to first assemble followed by the injection of NEM in panel A than when PFO^N197C is labeled with NEM prior to its injection into the liposomes in panel B.