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To determine how different constituents of pulmonary surfactant affect its phase behavior, we measured wide-angle x-ray scattering (WAXS) from oriented bilayers. Samples contained the nonpolar and phospholipids (N&PL) obtained from calf lung surfactant extract (CLSE), which also contains the hydrophobic surfactant proteins SP-B and SP-C. Mixtures with different ratios of N&PL and CLSE provided the same set of lipids with different amounts of the proteins. At 37°C, N&PL by itself forms coexisting Lα and Lβ phases. In the Lβ structure, the acyl chains of the phospholipids occupy an ordered array that has melted by 40°C. This behavior suggests that the Lβ composition is dominated by dipalmitoyl phosphatidylcholine (DPPC), which is the most prevalent component of CLSE. The Lβ chains, however, lack the tilt of the Lβ′ phase formed by pure DPPC. At 40°C, WAXS also detects an additional diffracted intensity, the location of which suggests a correlation among the phospholipid headgroups. The mixed samples of N&PL with CLSE show that increasing amounts of the proteins disrupt both the Lβ phase and the headgroup correlation. With physiological levels of the proteins in CLSE, both types of order are absent. These results with bilayers at physiological temperatures indicate that the hydrophobic surfactant proteins disrupt the ordered structures that have long been considered essential for the ability of pulmonary surfactant to sustain low surface tensions. They agree with prior fluorescence micrographic results from monomolecular films of CLSE, suggesting that at physiological temperatures, any ordered phase is likely to be absent or occupy a minimal interfacial area.

To establish how the hydrophobic surfactant proteins, SP-B and SP-C, promote adsorption of lipids to an air/water interface, we used x-ray diffuse scattering (XDS) to determine an order parameter of the lipid chains (S(xray)) and the bending modulus of the lipid bilayers (K(C)). Samples contained different amounts of the proteins with two sets of lipids. Dioleoylphosphatidylcholine (DOPC) provided a simple, well characterized model system. The nonpolar and phospholipids (N&PL) from extracted calf surfactant provided the biological mix of lipids. For both systems, the proteins produced changes in S(xray) that correlated well with K(C). The dose-response to the proteins, however, differed. Small amounts of protein generated large decreases in S(xray) and K(C) for DOPC that progressed monotonically. The changes for the surfactant lipids were erratic. Our studies then tested whether the proteins produced correlated effects on adsorption. Experiments measured the initial fall in surface tension during adsorption to a constant surface area, and then expansion of the interface during adsorption at a constant surface tension of 40 mN/m. The proteins produced a sigmoidal increase in the rate of adsorption at 40 mN/m for both lipids. The results correlated poorly with the changes in S(xray) and K(C) in both cases. Disordering of the lipid chains produced by the proteins, and the softening of the bilayers, fail to explain how the proteins promote adsorption of lipid vesicles.

To determine how different constituents of pulmonary surfactant affect its phase behavior, we measured wide-angle x-ray scattering (WAXS) from oriented bilayers. Samples contained the nonpolar and phospholipids (N&PL) obtained from calf lung surfactant extract (CLSE), which also contains the hydrophobic surfactant proteins SP-B and SP-C. Mixtures with different ratios of N&PL and CLSE provided the same set of lipids with different amounts of the proteins. At 37°C, N&PL by itself forms coexisting L(α) and L(β) phases. In the L(β) structure, the acyl chains of the phospholipids occupy an ordered array that has melted by 40°C. This behavior suggests that the L(β) composition is dominated by dipalmitoyl phosphatidylcholine (DPPC), which is the most prevalent component of CLSE. The L(β) chains, however, lack the tilt of the L(β′) phase formed by pure DPPC. At 40°C, WAXS also detects an additional diffracted intensity, the location of which suggests a correlation among the phospholipid headgroups. The mixed samples of N&PL with CLSE show that increasing amounts of the proteins disrupt both the L(β) phase and the headgroup correlation. With physiological levels of the proteins in CLSE, both types of order are absent. These results with bilayers at physiological temperatures indicate that the hydrophobic surfactant proteins disrupt the ordered structures that have long been considered essential for the ability of pulmonary surfactant to sustain low surface tensions. They agree with prior fluorescence micrographic results from monomolecular films of CLSE, suggesting that at physiological temperatures, any ordered phase is likely to be absent or occupy a minimal interfacial area.

The hydrophobic surfactant proteins, SP-B and SP-C, promote rapid adsorption by the surfactant lipids to the surface of the liquid that lines the alveolar air sacks of the lungs. To gain insights into the mechanisms of their function, we used X-ray diffuse scattering (XDS) and molecular dynamics (MD) simulations to determine the location of SP-B and SP-C within phospholipid bilayers. Initial samples contained the surfactant lipids from extracted calf surfactant with increasing doses of the proteins. XDS located protein density near the phospholipid headgroup and in the hydrocarbon core, presumed to be SP-B and SP-C, respectively. Measurements on dioleoylphosphatidylcholine (DOPC) with the proteins produced similar results. MD simulations of the proteins with DOPC provided molecular detail and allowed direct comparison of the experimental and simulated results. Simulations used conformations of SP-B based on other members of the saposin-like family, which form either open or closed V-shaped structures. For SP-C, the amino acid sequence suggests a partial α-helix. Simulations fit best with measurements of XDS for closed SP-B, which occurred at the membrane surface, and SP-C oriented along the hydrophobic interior. Our results provide the most definitive evidence yet concerning the location and orientation of the hydrophobic surfactant proteins.

To determine how different components affect the structure of pulmonary surfactant, we measured X-ray scattering by samples derived from calf surfactant. The surfactant phospholipids demonstrated the essential characteristics of the L(γ) phase: a unit cell with a lattice constant appropriate for two bilayers; crystalline chains detected by wide angle X-ray scattering (WAXS). The electron density profile, obtained from scattering by oriented films at different relative humidities (70–97%), showed that the two bilayers, arranged as mirror images, each contain two distinct leaflets with different thicknesses and profiles. The detailed structures suggest one ordered leaflet that would contain crystalline chains, and one disordered monolayer likely to contain the anionic compounds, which constitute ~10% of the surfactant phospholipids. The spacing and temperature-dependence detected by WAXS fit with an ordered leaflet composed of dipalmitoyl phosphatidylcholine. Physiological levels of cholesterol had no effect on this structure. Removing the anionic phospholipids prevented formation of the L(γ) phase. The cationic surfactant proteins inhibited L(γ) structures, but at levels unlikely related to charge. Because the L(γ) phase, if arranged properly, could produce a self-assembled ordered interfacial monolayer, the structure could have important functional consequences. Physiological levels of the proteins, however, inhibit formation of the L(γ) structures at high relative humidities, making their physiological significance uncertain.