Volume 114, Issue 5, Pages 1103-1115 (March 2018) Enhanced Ordering in Monolayers Containing Glycosphingolipids: Impact of Carbohydrate Structure  Erik B. Watkins, Shelli L. Frey, Eva Y. Chi, Kathleen D. Cao, Tadeusz Pacuszka, Jaroslaw Majewski, Ka Yee C. Lee  Biophysical Journal  Volume 114, Issue 5, Pages 1103-1115 (March 2018) DOI: 10.1016/j.bpj.2017.12.044 Copyright © 2018 Biophysical Society Terms and Conditions

Figure 1 Chemical structures of DPPC, CM, GA1, and GM1 molecules used in the studies. Biophysical Journal 2018 114, 1103-1115DOI: (10.1016/j.bpj.2017.12.044) Copyright © 2018 Biophysical Society Terms and Conditions

Figure 2 Out-of-plane structures of representative DPPC:GM1 and DPPC:GA1 monolayers at 30 mN/m. (A) XR data and fits for 75:25 DPPC:GM1 and 75:25 DPPC:GA1 mixtures and (B) 25:75 DPPC:GM1 and 25:75 DPPC:GA1 mixtures are compared to pure DPPC and pure GM1. Data (symbols) and fits (shaded regions) are divided by the Fresnel reflectivity and offset vertically for clarity. Electron density distributions matching the fits are shown in (C) and (D) as pairs of lines defining the boundary of accepted fits. The center of the headgroups is located at z = 0, below which is the tail region (z < 0) and above which is the headgroup region (z > 0). To see this figure in color, go online. Biophysical Journal 2018 114, 1103-1115DOI: (10.1016/j.bpj.2017.12.044) Copyright © 2018 Biophysical Society Terms and Conditions

Figure 3 GIXD Bragg peak profiles for varying compositions of binary (A) DPPC:CM, (B) DPPC:GA1, and (C) DPPC:GM1 monolayers. Solid (red online) lines show fits to the data with dotted (red online) lines corresponding to individual diffraction peak contributions to the total calculated fit. All data were collected using the same incident flux so absolute intensities between measurements may be compared. Data sets are shifted vertically, and intensity scales of each panel vary for clarity. Error bars based on Poisson counting statistics are shown and are typically comparable to the size of the data symbols. To see this figure in color, go online. Biophysical Journal 2018 114, 1103-1115DOI: (10.1016/j.bpj.2017.12.044) Copyright © 2018 Biophysical Society Terms and Conditions

Figure 4 Integrated diffracted intensity of GIXD from monolayers of DPPC:CM (diamonds; black online), DPPC:GA1 (circles; blue online), and DPPC:GM1 (squares; red online) at 30 mN/m and 30°C. The integrated intensity is correlated with the degree of lipid chain ordering in the monolayer and shows a maximum at ∼40 mol % GA1 and 10 mol % GM1. Dashed lines are presented as a visual guide. Error bars are based on Poisson counting statistics. To see this figure in color, go online. Biophysical Journal 2018 114, 1103-1115DOI: (10.1016/j.bpj.2017.12.044) Copyright © 2018 Biophysical Society Terms and Conditions

Figure 5 In-plane coherence length of ordered domains within monolayers of DPPC:CM (triangles; black online), DPPC:GA1 (circles; blue online), and DPPC:GM1 (squares; red online). Domain sizes larger than the pure components are observed for DPPC:GA1 and DPPC:GM1 mixtures. Dashed lines are presented as a visual guide. Error bars are based on χ2 + 1 change from Bragg peak fit parameter variation. To see this figure in color, go online. Biophysical Journal 2018 114, 1103-1115DOI: (10.1016/j.bpj.2017.12.044) Copyright © 2018 Biophysical Society Terms and Conditions

Figure 6 (A) Area per molecule within ordered domains obtained from GIXD and (B) average area per molecule obtained from isotherms for lipid packing in monolayers of DPPC:CM (diamonds and triangles; black online), DPPC:GA1 (circles; blue online), and DPPC:GM1 (squares; red online) at 30 mN/m (20,26). Downward pointing triangles correspond to distorted hexagonal phases and upward pointing triangles to hexagonal phases within the DPPC:CM monolayers. Deviation of the ordered domain APM from ideal mixing is shown (C) based on the APM of ordered domains of the pure components and (D) based on the average APM of the pure components. Due to lack of diffraction, the ordered APM of the pure GM1 monolayer was estimated by a linear extrapolation from the 80, 70, and 60 mol % GM1 monolayer APMs. Dashed lines are presented as a visual guide. Error bars are on based on error propagation from the Bragg peak fitting and an estimated 1 Å2 error in the isotherm APMs. To see this figure in color, go online. Biophysical Journal 2018 114, 1103-1115DOI: (10.1016/j.bpj.2017.12.044) Copyright © 2018 Biophysical Society Terms and Conditions

Figure 7 Electron density difference between pure DPPC and 75:25 DPPC:GM1 (upper two lines; red online) from Fig. 2 C shows GM1 headgroup position in regard to the center of DPPC headgroup (z = 0). The ρe− difference between 75:25 DPPC:GM1 and 75:25 DPPC:GA1 (lower two lines; black online) indicates sialic acid position. The width of the ribbons represents uncertainty in the distributions. To see this figure in color, go online. Biophysical Journal 2018 114, 1103-1115DOI: (10.1016/j.bpj.2017.12.044) Copyright © 2018 Biophysical Society Terms and Conditions