1. Volume 113, Issue 9, Pages 2016-2028 (November 2017)
Membrane Cholesterol Reduces Polymyxin B Nephrotoxicity in Renal Membrane Analogs 
Adree Khondker, Richard J. Alsop, Alexander Dhaliwal, Sokunthearath Saem, Jose M. Moran-Mirabal, Maikel C. Rheinstädter 
Biophysical Journal 
Volume 113, Issue 9, Pages (November 2017)
DOI: /j.bpj
Copyright © 2017 Biophysical Society Terms and Conditions

2. Figure 1
(a) Given here is molecular structure of the PmB and its cartoon representation. (b) This depicts the two prominent models of PmB action: the carpet and barrel-stave model. (c) Renal membrane analogs were created from PC, PE, and PS phospholipids with and without cholesterol as solid supported membrane stacks in the experiments. Analogous systems were prepared for the MD simulations. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

3. Figure 2
Two-dimensional x-ray data (a–h) were collected for all membrane complexes to study molecular structure perpendicular to the solid supported membranes (out-of-plane, qz) and parallel to the membranes (in-plane, q||). Sample type is denoted in the top-right corner. Right panels show high-resolution reciprocal space maps, with increased defect scattering with PmB. A cubic pattern is observed with added PmB. Intensities are shown on a logarithmic scale. (i) The volume fraction of cubic phase was estimated by integrating the cubic [211] signal and normalizing by integrating the same area in the absence of PmB. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

4. Figure 3
(a) Shown here are reflectivities of the four different testing conditions; the coexistence of the lamellar with a cubic lipid phase (Im3m) for states including PmB is shown in (b). The occurrence of this spongy phase is indicative of pore formation in the membrane stack. (c) Given here are the electron density profiles constructed from these peaks in cholesterol-depleted membranes and (d) in cholesterol-enriched membranes. Position of the PmB molecules can be determined from the difference curve shown in (e). The behavior of the lamellar width, dz, for all systems is shown in (f). The presence of PmB in the membranes leads to a decrease in dz. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

5. Figure 4
(a) Calculated electron density of the PmB molecules at different angles. Whereas the electron distribution is well fit by two Gaussian distributions at 0 and 45°, only one Gaussian is needed to describe the peptide oriented at 90°, parallel to the membranes. (b) Given here are difference curves Δρ fitted with the theoretical densities of PmB. (c) From fitting, two states are shown at θ = 80° external to the bilayer at position 1 and inserted into the bilayer at position 2 at θ = 55° in both systems. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

6. Figure 5
Snapshots of the MD Simulations in cholesterol-rich and cholesterol-depleted membranes with and without PmB. In (a) and (b), the PmB peptides were added to the hydration water layer and spontaneously attached to the membranes. Without cholesterol (a), the PmB molecules started to aggregate on the membranes and lead to an indentation and membrane thinning. In the presence of cholesterol in (b), no aggregation was observed and the membrane structure was overall intact. Systems are shown with periodic extensions with the simulation box highlighted. In (c) and (d), the PmB was added to the membrane core. Also here, PmB molecules start to cluster in the cholesterol-depleted membranes and led to significant distortion of the membranes and water intake. The subplot (inset) zooms into 3 PmB molecules interacting and forming clusters during 30 ns. In the presence of cholesterol, in (d), membrane stability was overall preserved and clustering of PmB was minimal after 100 ns. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

7. Figure 6
(a) Given here is the proportion of water molecules inside the bilayers, found between the headgroups, when PmB was added to water layer and inserted in the membranes. The highest amount of intramembrane water was found when PmB was inserted and when there was no cholesterol. (b) Diffusion constants of lipid and PmB molecules are given. In all cases, cholesterol was found to slow down lipid and peptide dynamics significantly. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

8. Figure 7
(a) Given here is a sketch of the cyclic voltammetry setup. Membranes were applied on a gold electrode. Voltage was swept at the working electrode and resulting current was monitored. (b) Given here are cyclic voltammograms in the absence of cholesterol. The insets plot the area of the scans, and indicate that the current recovers after 30 s. (c) With cholesterol, the current does not recover even after 5 min, indicating significantly reduced membrane damage. The scale bar corresponds to half the diameter of the gold surface. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

9. Figure 8
Basis of PmB damage in model membranes. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions