An Anionic Phospholipid Enables the Hydrophobic Surfactant Proteins to Alter Spontaneous Curvature

Abstract

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.

Hydrophobic Surfactant Proteins Strongly Induce Negative Curvature

“…In addition to introducing negative charge, DOPG also expands the H II cylinders (22,30). This observation suggests that DOPG might alter the effect of the proteins by changing the dimensions of the cylindrical monolayers rather than by affecting the charge.…”

“…The components of surfactant vesicles insert into the interface collectively (16)(17)(18). As with fusion, several compounds affect adsorption according to how they alter curvature (19)(20)(21)(22)(23). These results suggest that surfactant proteins might accelerate adsorption by facilitating the formation of a negatively curved, rate-limiting intermediate ( Fig.…”

“…1 B) analogous to the stalk proposed for fusion (24)(25)(26). Direct measurements with H II structures have shown that the surfactant proteins can promote more negative curvature, but their effect is limited, amounting to a change of only~8% (22).…”

“…In the H II phase formed by dioleoylphosphatidylethanolamine (DOPE), the proteins have no effect (22). The change in curvature produced by the proteins occurs with dioleoylphosphatidylglycerol (DOPG) added to DOPE (DOPE:DOPG, 9:1 mol/mol) to replicate the~10% content of anionic lipids in pulmonary surfactant (27).…”

“…SP-B and SP-C have a net charge of þ14 (28) and þ2-3 (29), respectively. Salt, however, which should screen electrostatic effects, produces a remarkably limited change (22).…”