Phase-Separation and Domain-Formation in Cholesterol-Sphingomyelin Mixture: Pulse- EPR Oxygen Probing  Laxman Mainali, Marija Raguz, Witold K. Subczynski  Biophysical Journal  Volume 101, Issue 4, Pages 837-846 (August 2011) DOI: 10.1016/j.bpj.2011.07.014 Copyright © 2011 Biophysical Society Terms and Conditions

Figure 1 Phase diagram of the Chol/ESM membrane. The broken line and solid circles indicate the temperature and Chol/ESM mixing ratios at which measurements were performed. Schematic drawings of membrane structures (including phases and domains) at different Chol/ESM mixing ratios ((a) 0; (b) 1:4; (c) 1:2; (d) 1:1; (e) 2:1; (f) 3:1) are presented. Biophysical Journal 2011 101, 837-846DOI: (10.1016/j.bpj.2011.07.014) Copyright © 2011 Biophysical Society Terms and Conditions

Figure 2 Representative SR signals with fitted curves and residuals (the experimental signal minus the fitted curve) of 7-PC obtained at different Chol/ESM mixing ratios (indicated in Fig. 1). Membrane specimens were equilibrated with nitrogen or a mixture of 50% air and 50% nitrogen. SR signals were satisfactorily fitted to a single-exponential function in the absence of molecular oxygen with time constants of (a) 3.56 ± 0.01 μs, (b) 3.78 ± 0.01 μs, (c) 4.12 ± 0.01 μs, (d) 4.59 ± 0.01 μs, (e) 4.62 ± 0.01 μs, and (f) 4.66 ± 0.01 μs. SR signals in the presence of molecular oxygen were fitted either to single exponentials with time constants of (a) 0.73 ± 0.01 μs, (c) 1.25 ± 0.01 μs, (d) 1.94 ± 0.01 μs, (e) 2.00 ± 0.01 μs, (f) 1.86 ± 0.01 μs, or double exponentials with time constants of (b) 1.19 ± 0.05 μs and 0.60 ± 0.06 μs (the middle residual in b is for single- and the lower residuals for double-exponential fits). Biophysical Journal 2011 101, 837-846DOI: (10.1016/j.bpj.2011.07.014) Copyright © 2011 Biophysical Society Terms and Conditions

Figure 3 Hydrophobicity around the nitroxide moiety of ASL and CSL in membranes made of Chol/ESM membranes plotted as a function of Chol/ESM mixing ratio. Biophysical Journal 2011 101, 837-846DOI: (10.1016/j.bpj.2011.07.014) Copyright © 2011 Biophysical Society Terms and Conditions

Figure 4 Representative SR signals with fitted curves and residuals for (A) ASL and (B and C) CSL in an ESM membrane with a Chol/ESM mixing ratio of 3. Signals were recorded for samples equilibrated with (A–C) 100% nitrogen, (A–B) with a gas mixture of 50% air and 50% nitrogen, and (C) in the presence of NiEDDA. For deoxygenated samples, SR signals were satisfactorily fit to a single-exponential function with time constants of (A) 3.00 ± 0.01 μs, (B) 3.41 ± 0.01 μs, and (C) 3.41 ± 0.01 μs (upper residuals are for single-exponential fit). The SR signal in the presence of molecular oxygen can be fitted satisfactorily with a single exponential function only for CSL with a time constant of (B) 2.12 ± 0.01 μs (lower residual is for single-exponential fit) and with double exponential curves for ASL with time constants of (A) 1.72 ± 0.07 μs and 0.48 ± 0.01 μs (the middle residual is for single- and the lower residual for double-exponential fits). The SR signal for CSL in the presence of NiEDDA can be fitted satisfactorily only with double-exponential curves with time constants of (C) 1.57 ± 0.04 μs and 0.74 ± 0.01 μs (the middle residual is for single- and the lower residual for double-exponential fits). Biophysical Journal 2011 101, 837-846DOI: (10.1016/j.bpj.2011.07.014) Copyright © 2011 Biophysical Society Terms and Conditions

Figure 5 (A) The OTP for ASL and CSL and (B) the NiEDDA accessibility parameter for CSL in Chol/ESM membranes plotted as a function of the Chol/ESM mixing ratio. Biophysical Journal 2011 101, 837-846DOI: (10.1016/j.bpj.2011.07.014) Copyright © 2011 Biophysical Society Terms and Conditions

Figure 6 Profiles of the OTP for Chol/ESM membranes obtained at Chol/ESM mixing ratios of (a) 0, (b) 1:4, (c) 1:2, (d) 1:1, (e) 2:1, and (f) 3:1 (schemes for membrane structures are shown in Fig. 1). Dotted lines indicate the OTP in the aqueous phase. Biophysical Journal 2011 101, 837-846DOI: (10.1016/j.bpj.2011.07.014) Copyright © 2011 Biophysical Society Terms and Conditions