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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.

Prior studies have shown that the biological mixture of the two hydrophobic surfactant proteins, SP-B and SP-C, produces faster adsorption of the surfactant lipids to an air/water interface, and that they induce 1-palmitoyl-2-oleoyl phosphatidylethanolamine (POPE) to form inverse bicontinuous cubic phases. Previous studies have shown that SP-B has a much greater effect than SP-C on adsorption. If the two proteins induce faster adsorption and formation of the bicontinuous structures by similar mechanisms, then they should also have different abilities to form the cubic phases. To test this hypothesis, we measured small-angle X-ray scattering on the individual proteins combined with POPE. SP-B replicated the dose-related ability of the combined proteins to induce the cubic phases at temperatures more than 25 °C below the point at which POPE alone forms the curved inverse-hexagonal phase. With SP-C, diffraction from cubic structures was either absent or present at very low intensities only with larger amounts of protein. The correlation between the structural effects of inducing curved structures and the functional effects on the rate of adsorption fits with the model in which SP-B promotes adsorption by facilitating formation of an inversely curved, rate-limiting structure.

Several small hydrophobic peptides can promote the fusion of lipid bilayers. The stalk model proposes that fusion proceeds via a negatively curved intermediate, and suggests that the peptides might achieve their fusogenic effect by enhancing negative spontaneous curvature of the individual lipid leaflets. Direct measurements on the unpaired monolayers of the inverse hexagonal (HII) phase have shown that the peptides can induce more negative curvature, but to a limited extent. The hydrophobic surfactant proteins, SP-B and SP-C, show similar behavior. They promote the adsorption of vesicles to an air/water interface by a process analogous to fusion. They enhance the negative spontaneous curvature of HII monolayers formed by dioleoyl phosphatidylethanolamine (DOPE):dioleoyl phosphatidylglycerol (9:1, mol:mol), but only by 8%. The studies here tested whether the limited response might reflect the size of the HII cylinder and the restricted space available for incorporation of the proteins. Small angle x-ray scattering showed that different ratios of DOPE with dioleoyl phosphatidylcholine (DOPC) produced HII cylinders with different radii. The effects of the proteins depended on the cylindrical size. Up to 3% (w:w) of the proteins produced no change in the smallest cylinders, formed by DOPE alone. With larger cylinders, the proteins reduced the radius. The magnitude of the change increased with cylindrical size. At 80% (mol:mol) DOPC, 5% (wt:wt) protein reduced the outer cylindrical radius by 78%. This change represented a 90% increase in the estimated magnitude of negative spontaneous curvature. These results show that demonstrating the ability of the hydrophobic surfactant proteins to promote negative curvature requires HII cylinders above a minimum size. Our findings raise the possibility that the effects on HII-curvature of other fusogenic peptides might also depend on the cylindrical radius. Acknowledgements: NIH; SSRL.

The rate at which surfactants lower surface tension (γ) during adsorption to an air/water interface normally slows progressively as γ falls. With vesicles of pulmonary surfactant, the slope of the γ-time isotherm initially decreases, but it then becomes steeper just before reaching the equilibrium γ. To determine the mechanisms that produce the accelerated drop in γ, we tested whether the steeper slopes reflected later time or lower γ. Adsorption at constant γ and to preexisting films showed that the steeper slope depended on γ rather than time, indicating that the acceleration resulted from changes in the film rather than in the adsorbing material. Compositional studies tested how removing cholesterol, the anionic phospholipids, and the hydrophobic proteins affected the late acceleration. Only the absence of the proteins eliminated the late adsorption. Vesicles of phospholipid with dioleoyl phosphatidylethanolamine, however, which, like the surfactant proteins, promotes fusion between vesicles, also demonstrated the accelerated late adsorption. These results suggest that the late acceleration occurs when vesicles that adsorb by fusion with the nascent film can interact more readily with a more densely packed monolayer. (Small angle x-ray diffraction studies conducted at the Stanford Synchrotron Radiation Lightsource.)

The hydrophobic surfactant proteins, SP-B and SP-C (SPs), promote rapid adsorption of phospholipids to an air-water interface by a mechanism that remains unclear. To understand the structural changes that lead to faster adsorption, we measured small-angle X-ray diffraction from dipalmitoylphosphatidylcholine (DPPC) bilayers containing varying concentration of SPs on solid supports. Swelling at different hydration allowed interpretation of the diffracted intensities and construction of the electron density profile. With increasing concentrations of protein (0 - 10 wt/wt %), the d-spacing and bilayer thickness increased. This change may reflect stretching of the hydrophobic region of the bilayer caused by the presence of the protein. Larger amounts of protein also flattened the electron density profiles, reducing the difference between minimum and maximum densities. A disordering effect of the proteins should cause greater thermal fluctuations of the bilayer, which would explain both the flattened density profile and the observed loss of higher order Bragg peaks.Figure 1: Electron density profiles of DPPC samples containing varying amount of SPs.View Large Image | View Hi-Res Image | Download PowerPoint SlideAcknowledgements: Stanford Synchrotron Radiation Lightsource (SSRL); NIH.

The hydrophobic surfactant proteins SP-B and SP-C greatly accelerate the adsorption of vesicles containing the surfactant lipids to form a film that lowers the surface tension of the air/water interface in the lungs. Pulmonary surfactant enters the interface by a process analogous to the fusion of two vesicles. As with fusion, several factors affect adsorption according to how they alter the curvature of lipid leaflets, suggesting that adsorption proceeds via a rate-limiting structure with negative curvature, in which the hydrophilic face of the phospholipid leaflets is concave. In the studies reported here, we tested whether the surfactant proteins might promote adsorption by inducing lipids to adopt a more negative curvature, closer to the configuration of the hypothetical intermediate. Our experiments used x-ray diffraction to determine how the proteins in their physiological ratio affect the radius of cylindrical monolayers in the negatively curved, inverse hexagonal phase. With binary mixtures of dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylcholine (DOPC), the proteins produced a dose-related effect on curvature that depended on the phospholipid composition. With DOPE alone, the proteins produced no change. With an increasing mol fraction of DOPC, the response to the proteins increased, reaching a maximum 50% reduction in cylindrical radius at 5% (w/w) protein. This change represented a doubling of curvature at the outer cylindrical surface. The change in spontaneous curvature, defined at approximately the level of the glycerol group, would be greater. Analysis of the results in terms of a Langmuir model for binding to a surface suggests that the effect of the lipids is consistent with a change in the maximum binding capacity. Our findings show that surfactant proteins can promote negative curvature, and support the possibility that they facilitate adsorption by that mechanism.

When compressed, alveolar films of pulmonary surfactant resist collapse from the air/water interface. This physiologically essential characteristic remains unexplained. Recent studies show that self-assembled films of pulmonary surfactant formed by adsorption are more resistant to collapse than spread monolayers containing the same constituents. One candidate for the self-assembled stable structure is the Lγ phase. The unit cell of this structure contains two lamellar bilayers. Half of the monolayers are ordered, and half are disordered.

The hydrophobic surfactant proteins, SP-B and SP-C, greatly accelerate the adsorption of the surfactant lipids to an air/water interface. Previous studies of factors that affect curvature suggest that vesicles may adsorb via a rate-limiting structure with prominent negative curvature, in which the hydrophilic face of the lipid leaflets is concave. To determine if SP-B and SP-C might promote adsorption by inducing negative curvature, we used small-angle x-ray scattering to test whether the physiological mixture of the two proteins affects the radius of cylindrical monolayers in the inverse hexagonal phase. With dioleoyl phosphatidylethanolamine alone, the proteins had no effect on the hexagonal lattice constant, suggesting that the proteins fail to insert into the cylindrical monolayers. The surfactant lipids also contain ∼10% anionic phospholipids, which might allow incorporation of the cationic proteins. With 10% of the anionic dioleoyl phosphatidylglycerol added to dioleoyl phosphatidylethanolamine, the proteins induced a dose-related decrease in the hexagonal lattice constant. At 30°C, the reduction reached a maximum of 8% relative to the lipids alone at ∼1% (w/w) protein. Variation of NaCl concentration tested whether the effect of the protein represented a strictly electrostatic effect that screening by electrolyte would eliminate. With concentrations up to 3 M NaCl, the dose-related change in the hexagonal lattice constant decreased but persisted. Measurements at different hydrations determined the location of the pivotal plane and proved that the change in the lattice constant produced by the proteins resulted from a shift in spontaneous curvature. These results provide the most direct evidence yet that the surfactant proteins can induce negative curvature in lipid leaflets. This finding supports the model in which the proteins promote adsorption by facilitating the formation of a negatively curved, rate-limiting structure.

Available evidence suggests that the hydrophobic surfactant proteins (SPs), SP-B and SP-C, accelerate adsorption of surfactant vesicles to an air/water interface by promoting formation of a negatively curved rate-limiting structure. In support of this model, the proteins induce several phosphatidylethanolamines to form inverse bicontinuous cubic phases, in which each leaflet has negative saddle-like curvature analogous to the hypothetical intermediate. The proteins could promote formation of cubic phases by changing spontaneous curvature (c0) of lipids, which would be reflected in the dimensions of the inverse hexagonal (HII) phase. With 1,2-dioleoyl phosphatidylethanolamine (DOPE), the SPs had no effect on the size of the HII phase, suggesting a constant c0. The study, however, lacked anionic phospholipids, which constitute ∼10% (mol:mol) of phospholipids in lung surfactant, and could engage in selective interactions with the cationic SPs. In this work, we used small-angle X-ray scattering to examine how SPs affect structures formed by DOPE mixed with 10% (mol:mol) anionic 1,2-dioleoyl phosphatidylglycerol (DOPG). With DOPG, the HII lattice-constant (a0) decreased in a dose-dependent manner with increasing levels of protein. This change could be caused by specific interactions between the SPs and DOPG, or nonspecific interactions between cationic SPs and the anionic phosphate group of DOPG. To determine whether the observed change in a0 was caused by nonspecific electrostatic effects, the measurements were repeated on samples prepared in buffered electrolyte. In the presence of counterions, the effect of SPs on a0 was significantly diminished. These results suggest that the change in the a0 for DOPE:DOPG in the absence of counterions is caused by nonspecific electrostatic interactions between the positively-charged proteins and anionic phospholipids, and are unlikely to play a major role in physiological media.Acknowledgements: Stanford Synchrotron Radiation Lightsource; NIH.