Structure of Supported Bilayers Composed of Lipopolysaccharides and Bacterial Phospholipids: Raft Formation and Implications for Bacterial Resistance  Jihong Tong, Thomas J. McIntosh  Biophysical Journal  Volume 86, Issue 6, Pages 3759-3771 (June 2004) DOI: 10.1529/biophysj.103.037507 Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 1 Schematic representation of the chemical structures of (A) an LPS molecule, (B) a PEI monomer, and (C) an LPS bilayer on a PEI-coated mica substrate. In (A) the lipid acyl chains of the lipid A moiety are represented by jagged lines and the sugar residues of the LPS polysaccharide cores and the O-specific chain are represented by hexagons. Rd LPS contains only the inner saccharide core, whereas the larger Ra LPS contains both the inner and outer saccharide cores. Biophysical Journal 2004 86, 3759-3771DOI: (10.1529/biophysj.103.037507) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 2 AFM images of Rd LPS bilayers on bare mica (A) scanned in water (fully hydrated condition) and (B) scanned in air (partially hydrated condition). Image size is 5μm2 and z-scale is 30nm. In all images lower surfaces are darker. For each image, a section analysis along the horizontal line in the image is shown below the image, with the arrow on the left pointing to the bare mica surface and the arrow on the right pointing to a typical Rd LPS region. From the section analysis the thickness of the Rd LPS is measured to be ∼7nm in each image. The Rd LPS bilayer coverage values are estimated to be 86% in water (A) and 36% in air (B). Biophysical Journal 2004 86, 3759-3771DOI: (10.1529/biophysj.103.037507) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 3 AFM image of PEI-coated mica surface, image size is 5μm2 and z-scale is 20nm. A central 1-μm2 defect was created by the AFM tip at contact mode with a high force load and a high scan speed. A section analysis along the line in the image (shown below the image) gives the thickness of the PEI layer as ∼1.2nm. Biophysical Journal 2004 86, 3759-3771DOI: (10.1529/biophysj.103.037507) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 4 AFM images of Rd LPS bilayers formed on PEI-coated mica (A) scanned in water and (B) scanned in air. Image size, 5μm2; z-scale, 30nm. For each image, a section analysis along the horizontal line in the image is shown below the image, with the arrow on the left pointing to the PEI-coated mica surface and the arrow on the right pointing to a typical LPS bilayer region. From the section analysis the thickness of the bilayer is measured to be ∼7nm in each image. The Rd LPS bilayer coverage values on PEI-coated mica are estimated to be 92% in water and 67% in air. The highest peaks in the section analysis of Rd LPS have a height of 14nm and probably correspond to a double bilayer region. Biophysical Journal 2004 86, 3759-3771DOI: (10.1529/biophysj.103.037507) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 5 AFM images of Ra LPS bilayers formed on PEI-coated mica by 4h SUV deposition at 60°C: (A) scanned in water and (B) scanned in air. Image size: 5×5μm, z-scale: 30nm. For each image, a section analysis along the horizontal line in the image is shown below the image, with the lower arrow pointing to the PEI-coated mica surface and the upper arrow pointing to a typical LPS bilayer region. From the section analysis the thickness of the bilayer is measured to be ∼9nm in each image. Biophysical Journal 2004 86, 3759-3771DOI: (10.1529/biophysj.103.037507) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 6 DSC thermograms of Rd LPS (3 mg/ml) and equimolar Rd LPS/BPL (4 mg/ml) MLVs. Note that the two thermograms are shown on different vertical scales. The arrow points in the direction of an endothermic transition. Biophysical Journal 2004 86, 3759-3771DOI: (10.1529/biophysj.103.037507) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 7 AFM images of (A) 1:1 Rd LPS/BPL and (B) 2:1 Rd LPS/BPL formed on PEI-coated mica scanned in water at 20°C. Image size, 2μm2; z-scale, 20nm. For each image, regions of two different thicknesses are observed. A section analysis along the horizontal line in the image is shown below the image, indicating that the difference in height of the two regions is ∼2nm. Biophysical Journal 2004 86, 3759-3771DOI: (10.1529/biophysj.103.037507) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 8 AFM images of (A) 1:1 Rd LPS/BPL and (B) 2:1 Rd LPS/BPL bilayers formed on PEI-coated mica, scanned in water at 40°C. Image size, 2μm2; z-scale, 20nm. For each image, a section analysis along the horizontal line in the image is shown below the image. Biophysical Journal 2004 86, 3759-3771DOI: (10.1529/biophysj.103.037507) Copyright © 2004 The Biophysical Society Terms and Conditions

Figure 9 Relationship between the predicted and observed BPL area in Rd LPS/BPL supported bilayers at 20°C and 40°C. Error bars represent standard deviations for three to five different AFM scans, and the dotted line is a least-squares fit to the 20°C values (R2=0.986). Biophysical Journal 2004 86, 3759-3771DOI: (10.1529/biophysj.103.037507) Copyright © 2004 The Biophysical Society Terms and Conditions