Supplemental Materials for “Decreasing Transmembrane Segment Length Greatly Decreases
Perfringolysin O Pore Size” by Qingqing Lin, Tong Wang, Huilin Li, and Erwin London
Supplemental Figure 1. SDS-AGE analysis of SDS-resistant oligomerization by PFO
mutants. The formation of oligomers was monitored by SDS-agarose electrophoresis. 10 μg
WT, long, or short PFO were incubated with 20 μl 6:4 (mol:mol) DOPC/cholesterol MLVs (25
mM lipids in PBS pH 5.1) for 1 h. The final pH was 6.0-6.7. Each sample was solubilized with
SDS sample buffer and separated by SDS-AGE.
Supplemental Figure 2. Time- and pH-dependent PFO-induced CB-dextran leakage.
Samples containing LUVs (~ 500 μM lipid) composed of 45:15:40 (mol:mol:mol)
DOPC/BrPC/cholesterol with 0.002 mol% Rho-DOPE entrapped MPTS (A-B) or CB-dextran 70
(C-D) without (control) or with 20 μg WT, 20 μg long or 30 μg short PFO were incubated for 1 h
or 2 h at room temperature. Then samples were then centrifuged at 84,000 g for 30 min at 4
degrees. Rhodamine (FRho) and cascade blue (FCB) emission intensity were both measured for the
pellet and supernatant. Fraction of CB-dextran in the supernatant = FCB-supernatant/(FCBsupernatant+FCB-pellet) – FRho-supernatant/(FRho-supernatant+FRho-pellet). (A) and (C) pH 7.4; (B) and (D) pH
5.3-5.7.
Supplemental Figure 1
Supplemental Figure 2
A
Fraction of MPTS in the supernatant
Control
Short
WT
Long
0.3
0.2
0.1
0.0
1
Fraction of CB-dextran in the supernatant
C
0.4
MTPS
neutral pH
0.5
hr
2
hr
CB-dextran 70,000, neutral pH
0.4
0.3
0.2
0.1
0.0
1
hr
2
hr
D
0.5
MTPS, low pH
0.4
0.3
0.2
0.1
0.0
1
Fraction of CB-dextran in the supernatant
Fraction of MPTS in the supernatant
B
0.5
0.5
hr
2
hr
CB-dextran 70,000, low pH
0.4
0.3
0.2
0.1
0.0
1
hr
2
hr